DNA encoding an avian E. coli iss

ABSTRACT

A nucleic acid sequence encoding an avian E. coli iss gene and an Iss polypeptide encoded thereby are disclosed. Methods for detecting and using such sequences are also provided as are immunogenic compositions and vaccines.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No.1P20RR11817-01, awarded by the NIH. The U.S. Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Pathogenic Escherichia coli ("E. coli") is the causative agent of avariety of diseases in humans and animals, and often have the ability toavoid, resist or inactivate a multi-cellular organisms chemical andcellular defenses for a significant period of time after the host hasbeen exposed to the pathogen. E. coli infection of the avian respiratorytract causes respiratory tract lesions and septicemia.

E. coli infections are often secondary to infections of birds byinfectious bronchitis virus, Newcastle disease virus and Mycoplasma spp(Gross, Disease of Poultry, 8th ed., 270-278 (1984)). Three componentspresent on the cell surface of E. coli assist in promoting the survivalof E. coli from serum and phagocytosis (Timmis et al., Current Topics inMicro. and Immunol., 118:197-218 (1985)). These three components are theacidic polysaccharide capsules, O-antigen lipopolysaccharide("O-antigens") and outer membrane proteins ("OMPs"). Some types ofO-antigens have been shown to mediate resistance to complement and toincrease bacterial virulence (Moll et al., FEMS Lett., 6:273-276 (1979);Pluschke et al., Infect. Immun., 42:907-913 (1983); and Goldman et al.,J. Bacteriol., 159:877-882 (1984)). Avian E. coli strains with one ofthree O-antigens, O1, O2 or O3, cause the majority of all E.coli-induced septicemic colibacillosis in birds (Naveh et al., AvianDisease, 28:651-661 (1984)). However, many other serotypes such asO78:K80 are also observed.

Septicemic colibacillosis occurs most commonly in 5 to 12-week oldbroiler chickens, but also occurs in newly hatched chicks and turkeypoults. The pathogenicity of E. coli for poultry has been correlatedwith various factors (Sussman, The Virulence of Escherichia coli,Academic Press Inc., Ltd., London (1985)). These factors includeantimicrobial resistance (Cloud et al., Avian Dis., 29:1084-1093(1985)); production of colicins, siderophores, type I pili andhemolysins (Arp et al., Avian Dis., 24:153-161 (1980)); resistance tohost complement (Ike et al., J. Vet. Med. Sci., 54:1091-1098 (1992));presence of certain plasmids (Binns et al., Nature, 279:778-781 (1979));motility (Wooley et al., Avian Dis., 6:679-684 (1992)); serotype, e.g.,O1, O2, O3 and O78 (Rosenberger et al., Avian Dis., 29:1094-1107(1985)); and invasiveness (Vidotto et al., Avian Dis., 34:531-538(1990)). Recent reports have shown that the ability of avian E. coli toresist the lytic effects of host complement is a major factor in thedevelopment of colibacillosis in poultry (Nolan et al., Avian Dis.,38:146-150 (1994); Nolan et al., Avian Dis., 3:395-397 (1992); Nolan etal., Avian Dis., 36:398-402 (1992)).

Two well-defined, interacting components that constitute the majorfirst-line of host defenses against invading bacteria are the complementsystem and phagocytosis (Miims, C. A., The Pathogenesis of InfectiousDisease, 2nd edn. Academic, London (1982); Taylor, P. W., Microbiol.Rev., 47:46-83 (1983)). Phagocytosis involves the ingestion ofparticulate material, including whole pathogenic microorganisms. Inphagocytosis, the plasma membrane expands around the particulatematerial to form phagosomes. Only specialized cells, phagocytes, areinvolved in phagocytosis and include such cells as blood monocytes,neutrophils and tissue macrophages.

Complement resistance of E. coli has generally been reported as relatedto several potential structural factors including a K1-antigenic capsule(Aquero et al., Infect. Immun., 40:359-368 (1984)), or other capsuletype (Russo et al., Infect. Immun, 61:3578-3582 (1993)), a smoothlipopolysaccharide (LPS) layer (Cross et al., In: Bacteria, Complementand the Phagocytic Cell, Vol. H24, F. C. Cabello and C. Pruzzo, eds.,Springer-Verlag, Berlin, pp. 319-334 (1988)), and certain OMPs includingTraT (Montenegro et al., J. Gen. Microbiol. 131:1511-1521 (1985); Mollet al., Infect. Immun. 28:359-367 (1980)), Iss (Binns et al., Infect.Immun. 35:654-659 (1982); Chuba et al., Mol. Gen. Genet., 216:287-192(1989)), and OmpA (Weiser et al., Infect. Immun. 52:2252-2258 (1991)).The absence of capsule as a complement-resistance mechanism indisease-associated avian E. coli isolates suggests that such isolatesmust employ other means to avoid the killing effects of complement.

Iss is an OMP encoded by an avian E. coli iss gene. Some reports haveindicated that E. coli isolates from avian subjects with a septicemicdisease are much more likely to have iss-related sequences than are E.coli isolates of apparently healthy birds. However, no significantdifference in the distribution of traT-related sequences (traT encodesthe OMP TraT) has been found in the same isolates. The plasmid locationof iss and traT suggests that their presence in different isolates mightbe more variable than the chromosomally-located ompA gene (Weiser etal., Infect. Immun., 59:2252-2258 (1991)).

The expression of a human E. coli iss gene was found to increase thevirulence of E. coli containing the gene by 100-fold for one-day oldchicks and to increase the complement resistance of transformed cellsover 20-fold for one-day old chicks (Binns et al., Infect. Immun.,35:654-659 (1982)). Genetic evidence suggests that iss obtained fromhuman E. coli is a derivative of a lambda gene known as bor (Barondesset al., J. Bacteriol., 177:1247-1253 (1995); Barondess et al., Nature,346:871-874; (1990); Chuba et al., Mol. Gen. Genet., 216:287-292(1989)). Bor is a lipoprotein of the cell envelope of E. coli lambdalysogens and appears to confer serum resistance on these lysogens.(Barondess et al., J. Bacteriol., 177:1247-1253 (1995)). The highsequence identity between the Bor polypeptide and an avian Isspolypeptide suggests that Iss is also targeted to the outer membrane.

Septicemic colibacillosis is an economically devastating problem for thepoultry industry in the United States. It is the most costly bacterialdisease of production poultry animals causing multi-million dollarlosses by the poultry industry annually. Control of this disease ishampered by the lack of a basic understanding about the virulencemechanisms employed by avian E. coli. For example, no singleidentifiable virulence marker has been associated with disease-causingavian E. coli. In other animal species, such as cattle, certain markersof virulence have been identified, and these markers have been used tofacilitate epidemiologic studies and to develop control strategiesdesigned to decrease the detrimental impact of colibacillosis on animalagriculture (Butler et al., G. L. Gyles ed. CAB International,Wallingford, UK:91-116 (1994)).

Therefore, a need exists to identify genes and other markers associatedwith complement-resistance of E. coli in birds. These genes can finctionas markers for disease-causing avian E. coli and the detection of thesegenes may form the basis for improved diagnostic and control strategiesfor avian colibacillosis, in addition to the formulation and preparationof immunogenic compositions useful to prevent or inhibit aviansepticemic diseases.

SUMMARY OF THE INVENTION

iss has been identified as a marker for avian E. coli virulence andcomplement-resistance, and is associated with septicemic disease inbirds. As used herein, the term iss or iss "gene" refers to a nucleicacid sequence or molecule that encodes an avian E. coli Iss polypeptide.Thus, the present invention provides an isolated and purified nucleicacid molecule that encodes an avian E. coli Iss polypeptide, abiologically active variant or subunit thereof. However, as describedmore fully below, an "Iss polypeptide" also includes an Iss "fusion"polypeptide.

A biologically active Iss polypeptide, variant or subunit thereof filingwithin the scope of the invention has at least about 1%, preferably atleast about 10%, more preferably at least about 50%, and even morepreferably at least about 90%, the activity of the polypeptidecomprising SEQ ID NO:2. A preferred nucleic acid sequence is a DNAmolecule encoding an Iss polypeptide (SEQ ID NO:2).

Another preferred embodiment of the invention is an isolated nucleicacid molecule comprising a nucleotide sequence as shown in FIG. 1 (SEQID NO:22). Biological samples from an avian subject suspected of beingexposed to, a carrier of, or afflicted with a septicemic disease, suchas colibacillosis, can be analyzed for the presence of an iss nucleicacid sequence or an Iss polypeptide sequence encoded thereby. The term"septicemia" or "septicemic disease" includes, but is not limited to,several avian diseases such as air-sacculitis, pneumonitis, septicemiccolibacillosis and colisepticemia.

The present invention further provides an expression cassette comprisinga preselected iss nucleic acid sequence operably linked to a promoterand functional in a host cell wherein said nucleic acid sequence encodesan Iss polypeptide, a biologically active variant or subunit thereof.The expression cassettes can be placed into expression vectors. Theserecombinant vectors can then be employed to transform prokaryotic andeukaryotic host cells. The expression of the preselected nucleic acidsequence in the transformed cell results in the production ofrecombinant Iss polypeptide or an Iss fuision polypeptide depending onthe selected vector. As used herein, an Iss "fusion" polypeptide orprotein is a product of a first preselected nucleic acid sequence, forexample a preselected iss sequence, and a second nucleic acid sequence,for example a glutathione S-transferase sequence, operably linked ateither the carboxyl terminal or amino terminal of the first sequence.The expression of this chimeric gene results in a single or continuouspolypeptide or protein when expressed and isolated from a host cell. Theproduct of this expression can enhance properties relating to, forexample, purification, isolation, targeting and increasedimmunogenicity.

The present invention further provides a method for detecting a nucleicacid sequence encoding an avian E. coli Iss polypeptide comprising (a)contacting an amount of an avian E. coli DNA from a biological samplefrom a bird at risk of, or afflicted with, a septicemic disease, with anamount of at least two oligonucleofides under conditions effective toamplify the DNA so as to yield an amount of an amplified DNA sequence,wherein at least one oligonucleotide is specific for the nucleic acid ofan avian E. coli iss nucleic acid sequence; and (b) detecting ordetermining the presence of the amplified DNA, wherein the presence ofthe amplified DNA is indicative of a bird at risk of, or afflicted with,a septicemic disease.

In yet another embodiment, a method of detecting nucleic acid moleculesencoding an avian E. coli iss gene in a biological sample is providedcomprising the steps of (a) introducing an avian E. coli iss specificoligonucleotide sequence and an iss non-specific oligonucleotidesequence into a biological sample suspected of containing an avian E.coli iss gene under conditions that permit the oligonucleotide sequencesto hybridize to the avian E. coli iss gene, and (b) amplifying thehybridized sequences by polymerase chain reaction to yield anamplification product.

Techniques of nucleic acid detection include DNA and RNA amplificationmethods and includes such methods as polymerase chain reaction ("PCR").Additionally, the detection of antibody responses specific for an Isspolypeptide encoded by an iss sequence can be used, for example, inELISA-based immunoassays for the serodiagnosis of an avian subjectsuspected of being exposed to, a carrier of, or afflicted with asepticemic disease. The presence or amount of an iss or an Iss sequencein a sample can then be compared to a control sample, e.g., an aviansubject known to be disease free.

Nucleic acid sequences of the invention can be produced by nucleic acidamplification techniques using novel oligonucleotide primers, such as,SEQ ID NOS:3, 4, 11, 12, 13, 14 and 15 employed in the synthesis. Thesenovel primers can also used as probes and in PCR to detect otherhomologous iss nucleic acid sequences. An oligonucleotide or primer ofthe invention preferably has at least about 70%, more preferably atleast about 85%, and most preferably at least about 90%, sequenceidentity or homology to SEQ ID NO:1.

The present invention also provides a polypeptide that can be used as acapture antigen to bind anti-polypeptide or anti-Iss antibodies in abiological sample obtained from an avian subject. For example, abiological sample comprising antibodies can be mixed with a purified Isspolypeptide to yield an antigen-antibody complex. The antibodies thatare bound to the antigen are separated from the antibodies that are notbound to the antigen. The antigen-antibody complex is then detected ordetermined.

The invention further provides a method for detecting or determining thepresence of Iss antibodies in a biological sample obtained from an aviansubject. The method comprises contacting an amount of a purified Isspolypeptide, variant or subunit thereof with the biological sample thatis suspected of comprising antibodies specific for the polypeptide for asufficient time to form complexes between at least a portion of theantibodies and a portion of the purified polypeptide. The presence oramount of the complexes is then determined or detected.

In another embodiment of the present invention, monoclonal andpolyclonal antibodies directed against an avian E. coli Iss polypeptideor fuision polypeptide are prepared. The recombinant polypeptide andantibodies are useful in the development of assays to detectIss-producing E. coli.

In yet another embodiment of the invention, an avian Iss polypeptide,variant or subunit thereof can be used to produce populations ofantibodies that, in turn can be used as the basis for assays to detectand quantify Iss polypeptide (or "protein") in samples derived from anavian subject. Also provided are populations of monoclonal andpolyclonal antibodies that specifically bind to an avian E. coli Isspolypeptide or GST-Iss fusion protein. These antibodies can also be usedin affinity chromatography, to purify avian Iss from natural sources.

Also provided is a diagnostic kit for detecting or determiningantibodies that specifically react to an avian E. coli Iss polypeptide,variant or subunit thereof. The kit comprises packaging, containing,separately packaged, a solid phase capable of binding a polypeptide anda known amount of a purified Iss polypeptide. Preferably, thepolypeptide has an amino acid sequence comprising SEQ ID NO:2, abiologically active variant or biologically active subunit thereof.

In another embodiment, an isolated and purified avian E. coli Isspolypeptide can be employed as a component in a diagnostic assay for"native" Iss in samples derived from avian biological samples. Forexample, polypeptide can be bound to a detectable label and employed ina competitive immunoassay for Iss protein.

Another preferred embodiment of the invention provides an immunogeniccomposition or a vaccine comprising an isolated and purified avian E.coli Iss polypeptide wherein the administration of the immunogeniccomposition or vaccine to an avian subject induces the production ofantibodies to said polypeptide. The produced antibodies can inhibit orblock subsequent infection of the host by a complement resistant and/orvirulent avian E. coli. Preferably, the immunogenic composition or avaccine is administered in combination with a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows partial alignment of the DNA sequence of the iss geneobtained from a virulent, complement-resistant avian E. coli isolate(102iss; SEQ ID NO:22), the DNA sequence of an iss gene from asepticemic human E. coli isolate (Eciss; SEQ ID NO. 5), and the DNAsequence of the lambda bor gene (lambor; SEQ ID NO.6).

FIG. 2 shows alignment of the predicted amino acid sequence of Iss froma virulent, complement-resistant avian E. coil (102 Iss; SEQ ID NO.2);Iss from a septicemic human E. coli isolate (Iss₋₋ Ec; SEQ ID NO.7), andthe lambda Bor protein (lamBor; SEQ ID NO.8).

FIG. 3 shows the iss gene sequence (SEQ ID NO:22) cloned in frame intoexpression vector pGEX-6P-3 including the PRESCISSION protease cleavagesite (SEQ ID NO:21). The polypeptide encoded by SEQ ID NO:21, designatedSEQ ID NO:20, is also shown. The GST fuision sequence, not shown, islocated upstream of the PRESCISSION protease cleavage site. Theamplified iss gene was cloned into the BamHI and the EcoRI sites of thepGEX-6P-3 vector.

FIG. 4 shows an SDS-PAGE of crude total protein preparations of aprotease-deficient E. coli containing the expression vector, pGEX-6P-3,or pGEX-6P-3 comprising the iss gene sequence, in the "uninduced" or"induced" state. Lane S=molecular weight standard in kD (PharmaciaBiotech Inc., Piscataway, N.J.); Lane 1U=pGEX-6P-3 alone in the"uninduced" state; Lane 3U=pGEX-6P-3 comprising iss in the "uninduced"state; Lane 1I=pGEX-6P-3 alone in the "induced" state; and lane3I=pGEX-6P-3 comprising iss in the "induced" state. Note that lane 3Ishows a 37 kD protein band not present in lanes 1U, 3U or 1I which isconsistent with an GST-Iss fuision polypeptide product.

FIG. 5 shows the crude total protein preparation of a protease-deficientE. coli, containing pGEX-6P-3 comprising the iss gene sequence. Totalprotein was prepared 3 hours post-induction of expression with IPTG.After resolution by SDS-PAGE, the proteins were transferred to PVDF andthe blot probed with anti-GST. Lane S=molecular weight standard in kD;Lanes 1, 2 and 3 =contain the same amount of crude bacterial lysate butdifferent concentrations of anti-GST (e.g, base solution of 5/mg/mldiluted to 1:500 (lane 3), 1:1000 (lane 2) and 1:2000 (lane 1)). In asecond PVDF blot using crude lysates of uninduced bacteria that wasprobed in a technique as described above, no bands were recognized (notshown), thus confirming the 37-kD band present in induced bacteria isthe GST-Iss fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

The identification and association of iss and Iss with clinical disease,e.g., septicemia, in birds due to virulent and complement resistant E.coli has been established. The data obtained from the isolation andcharacterization of iss and Iss sequences has clarified thedisease-causing potential of isolates that carry the iss gene, andestablished the importance of Iss to avian E. coli virulence andcomplement resistance. Moreover, iss and Iss employed as markers enablethe development of diagnostic procedures for use in avian colibacillosisoutbreaks and epidemiological studies and provide development of suchdiagnostic procedures by production of, for example, monoclonalantibodies to Iss.

Definitions

In describing the present invention, the following terminology will beused in accordance with the definitions set out below.

The terms "isolated and/or purified," refer to in vitro preparation,isolation and/or purification of a nucleic acid molecule, polypeptide orpeptide of the invention, so that it is not associated with in vivosubstances.

As used herein, an "Iss polypeptide or amino acid sequence" ispreferably an avian E. coli iss derived polypeptide having an amino acidsequence comprising SEQ ID NO:2, or a biologically active variant orsubunit thereof. A "variant" Iss polypeptide is a polypeptide having anamino acid sequence that has at least about 87%, preferably at leastabout 90%, and more preferably at least about 95%, but less that 100%,sequence identity or homology to SEQ ID NO:2 or a biologically activesubunit thereof. A variant polypeptide, peptide or amino acid sequenceof the invention may include amino acid residues not present in thecorresponding wild type Iss polypeptide or peptide, as well as internaldeletions relative to the corresponding wild type Iss polypeptide. Asused herein, a "subunit" is a biologically active portion, region orpeptide of a full-length Iss polypeptide, e.g., SEQ ID NO:2.

Preferably, the polypeptides and peptides of the instant invention arebiologically active. For example, biologically active Iss polypeptides,peptides and variants thereof falling within the scope of the inventionhave at least about 1%, preferably at least about 10%, more preferablyat least about 50%, and even more preferably at least about 90%, theactivity of the polypeptide comprising SEQ ID NO:2. The activity of anIss polypeptide or peptide, can be measured by methods well known to theart including, but not limited to, the ability of the polypeptide,peptide to be bound by antibodies specific for Iss (e.g., specific forthe Iss protein) or the ability of the polypeptide or peptide to elicita sequence-specific immunologic response when the polypeptide isadministered to an animal such as an avian subject. An avian E. coli Isspolypeptide, peptide, variant, subunit or fragment thereof is"immunologically reactive" with an antibody when it binds to an antibodydue to antibody recognition of a specific epitope contained within thepolypeptide. Immunological reactivity may be determined by antibodybinding, more particularly by the kinetics of antibody binding, and/orby competition in binding using as competitor(s) a known polypeptide(s)containing an epitope against which the antibody is directed. Thetechniques for determining whether a polypeptide is immunologicallyreactive with an antibody are known in the art.

Preferably, the immunologic response is a humoral response, i.e,antibody response, directed to a particular epitope on the polypeptideor peptide. More preferably, the presence of antibodies specific for anIss epitope correlates with a septicemic infection in an avian subject.As used herein, "epitope" refers to an antigenic determinant of apolypeptide. An epitope could comprise 3 amino acids in a spatialconformation which is unique to the epitope. Generally an epitopeconsists of at least 5 such amino acids, and more usually, consists ofat least 8-10 such amino acids. Methods of determining the spatialconformation of amino acids are known in the art, and include, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance.

An isolated "variant" nucleic acid sequence of the present invention isa nucleic acid sequence that has at least 87%, preferably at least about90%, and most preferably at least about 95%, but less than 100%, nucleicacid sequence identity or homology to a the nucleotide sequence of thecorresponding wild type nucleic acid molecule, e.g., a DNA sequencecomprising SEQ ID NO:22. However, a variant nucleic acid molecule of theinvention may include nucleotide bases not present in the correspondingwild type nucleic acid molecule, as well as internal deletions relativeto the corresponding wild type nucleic acid molecule. As used herein anucleic acid "subunit" is a biologically active portion or region of afull-length iss nucleic acid sequence, e.g., SEQ ID NO:22.

The term "hybridization under stringent conditions" refers to thoseconditions that (1) employ low ionic strength and high temperature forwashing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1%sodium lauryl sulfate at about 68° C., or (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNacl, 75 mM sodium citrate at 42° C. Another example is use of 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

As used herein, the term "target region" refers to a region of thenucleic acid which is to be amplified and/or detected.

A "genomic clone" refers to a DNA fragment derived directly fromcellular DNA rather than from messenger RNA, the usual source for cDNAclones. Genomic and cDNA clones have different sequences due to RNAsplicing.

BACKGROUND

The general techniques used in cloning avian E. coli iss nucleic acidsequences (e.g., PCR technology, sequencing nucleic acid clones andpolypeptides derived therefrom, constructing expression vectors orexpression cassettes, transforming cells, performing immunologicalassays such as radioimmunoassays and ELISA assays, and growing cells inculture) are known in the art and laboratory manuals are availabledescribing these techniques. See, for example, Sambrook, Fitsch &Maniatis, Molecular Cloning; A Laboratory Manual, Cold Spring HarborLaboratory Press (1989); DNA Cloning, Volumes I and II (D. N. Glover ed.1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal CellCulture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRLPress, 1986); B. Perbal; A Practical Guide to Molecular Cloning (1984);the Series, Methods in Enzymology (Academic Press, Inc.); ImmunochemicalMethods in Cell and Molecular Biology (Academic Press, London), Scopes,(1987), Protein Purification: Principles and Practice, Second Edition(Springer-Verlag, N.Y.), and Handbook of Experimental Immunology,Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

I. Identification of Isolates Falling Within the Scope of the Invention

A. Sources of Avian E. coli Isolates

E. coli iss isolates have been collected from a variety of aviansubjects (e.g., turkeys, chickens and ducks) clinically diagnosed with asepticemic disease or exposed to other avians having, afflicted with, orsuspected of a septicemic disease. Biological samples have been obtainedfrom sources such as tissues, organs, blood, serum, bone and yolk sacs.As used herein, the term "biological sample" refers to material derivedfrom, obtained from, or collected from the aforementioned sources of ananimal. Preferably, the biological sample is from an avian subject.Other sources of nucleic acid sequences encoding avian E. coli Isspolypeptides can be derived from any eukaryotic source, preferably anavian farm animal, known or believed to be naturally or experimentallyinfected by a virulent, septicemic causing avian E. coli.

A phenotypic comparison of 40 avian E. coli iss isolates from theintestines of normal chickens and 40 avian E. coli iss isolates from theorgans of colisepticemic chickens was performed. The isolates fromcolisepticemic chickens were shown to be more likely to producesiderophores and type I pili, and exhibit greater complement resistance,than were the intestinal isolates from normal chickens. A closerexamination of the 10 most complement-resistant colisepticemic issisolates and the 10 most complement-sensitive intestinal iss isolatesrevealed that an iss isolate's ability to resist complement directlycorrelated with an isolates ability to kill chick embryos (Wooley etal., Avian Dis., 36:679-684 (1992)).

The lytic activity of complement on test isolates was determined by aquantitative microtiter test method (Lee et al., Avian Dis. 35:892-896(1991)). The quantitative microtiter test results were analyzed bycalculating the regression coefficients (β) of the slopes of growth overtime. The β values were ranked and compared using a Dunnett's test(α=0.05). These rankings permitted the classification an E. coli aseither sensitive, intermediate or resistant to the action of chickencomplement. This observation was confirmed by mutational analysis.Specifically, a mutant E. coli differing from a virulent wild-type E.coli in the single factor of complement resistance was prepared andtested (Nolan et al., Avian Dis., 36:398-402 (1992)). Characterizationof this complement-sensitive mutant revealed that it was less virulentthan the wild-type isolate (Kottom et al., Avian Dis. (1997); Nolan etal., Avian Dis., 38:146-150 (1994); Nolan et al., Avian Dis., 36:398-402(1992)). This attribute provides further evidence that complementresistance is an important contributor in the observed virulence ofavian E. coli.

The occurrence of iss related sequences among clinical human and avianE. coli isolates is very informative. For example, Femendez-Beros etal., J. Clin. Micro., 28:742-746 (1990) examined 200 E. coli isolatesfrom patients with diarrhea and 146 isolates from patients withbacteremia for their possession of several virulence-related sequencesincluding traT and iss. No difference was found between the two groupsin distribution of these two genes. The iss-related sequences occurredin about 25-28% of all isolates examined, and the traT-related sequencesoccurred in 41-44% of these same isolates.

In the present invention, 210 isolates, derived from a variety ofsources, such as tissue, bone, blood, serum and yolk sacs of birds withcolibacillosis and 56 fecal isolates of healthy birds were examined. Itwas found that 76% of the isolates of sick chickens containediss-related sequences, and only 23% of the isolates of healthy chickenscontained such sequences. There was no significant difference in thedistribution of traT-related sequences between the two groups,suggesting that traT was not an important virulence factor for avian E.coli isolates. Conversely, the large difference in the distribution ofiss among isolates of birds suggests iss was a potentially importantvirulence factor for avian E. coli. Additionally, the difference indistribution of iss-related sequences in isolates from birds and humanswith extraintestinal colibacillosis suggested that iss was potentiallymore important for complement resistance and virulence with avian E.coli than with human E. coli.

The differences in the distribution of iss and capsule, a polysaccharidelayer located outside the cell wall of bacteria, among avian and humanE. coli isolates further emphasize the intriguing differences betweenavian and mammalian colibacillosis and suggest that the avian model ofcolibacillosis is a unique system in which to observe complementresistance. The uniqueness of this system reflects the nature ofcolibacillosis in birds, the entry point of E. coli into a host bird,and the initial sites of colonization of the host. As mentionedpreviously, the route of entry for most forms of avian colibacillosis isthe respiratory tract. Enteric forms of avian colibacillosis are rare(Gyles, In: Pathogenesis of Bacterial Infections in Animals, 2nd ed., C.L. Gyles and C. O. Thoen, eds., Iowa State University Press, Ames, IA,pp. 164-187 (1993)). This is not the case in mammals, where intestinalforms of colibacillosis are common (Gyles, In: Pathogenesis of BacterialInfections in Animals, 2nd ed., C. L. Gyles and C. O. Thoen, eds., IowaState University Press, Ames, IA, pp. 164-187 (1993)). Moreover, theentry point taken by most disease-causing E. coli of avians is primarilythrough the respiratory tract, unlike mammals, wherein the route ofinitial entry is likely to be the gastrointestinal tract (Gyles, In:Pathogenesis of Bacterial Infections in Animals, 2nd ed., C. L. Gylesand C. O. Thoen, eds., Iowa State University Press, Ames, IA, pp.164-187 (1993)).

B. Isolation of Nucleic Acid Molecules of the Invention

A nucleic acid molecule encoding an Iss polypeptide can be identifiedand isolated using standard methods, as described by Sambrook et al.,(1989). For example, polymerase chain reaction can be employed toisolate and clone iss genes. "Polymerase chain reaction" or "PCR" refersto a procedure or technique wherein amounts of a preselected piece ofnucleic acid, RNA and/or DNA, are amplified as described in U.S. Pat.No. 4,683,195. Generally, sequence information from the ends of theregion of interest or beyond is employed to design oligonucleotideprimers. These primers will be identical or similar in sequence toopposite or complimentary strands of the template to be amplified. PCRcan be used to amplify specific RNA sequences, specific DNA sequencesfrom total genomic DNA, and cDNA transcribed from total cellular RNA,bacteriophage or plasmid sequences and the like, to yield anamplification product. See also, Mullis et al., Cold Harbor Symp. Ouant.Biol., 51:263 (1987); Erlich, ed., PCR Technology (Stockton Press, NY,1989).

iss nucleic acid sequences can be isolated from septicemic bird tissuesamples by PCR techniques employing iss specific oligonucleotideprimers, for example, SEQ ID NO.3 ,4, 11, 12, 13, 14, and 15. Theseprimers will be identical or similar in sequence to opposite strands ofthe template to be amplified. Primers are made to correspond to nucleicacid molecules encoding highly conserved regions of polypeptides.However, these nucleic acid molecules can be introduced into expressioncassettes, which, when expressed in a host, can provide "anti-sense"nucleic acid transcripts. Thus, the present invention also providesisolated and purified "antisense" nucleic acid molecules that have atleast about 80%, preferably about 90%, and most preferably at leastabout 98%, nucleotide sequence complementary to SEQ ID NOs.1, 11, 12,13, 14 and 15.

Alternatively, nucleotide sequences can be obtained and prepared by asequence comparison of other related genes, such as the traT and ompAsequences. Preferably, at least two primers are prepared, one of whichis predicted to anneal to the antisense strand, and the other of whichis predicted to anneal to the sense strand of a nucleic acid moleculethat encodes the avian E. coli Iss polypeptide. The products of each PCRreaction are separated by agarose gel electrophoresis, and theconsistently amplified products are purified and cloned directly into asuitable vector such as a plasmid vector. Products obtained therefromcan be sequenced manually using standard procedures or with an automatedsequencer, for example, LICOR™.

Alternatively, DNA libraries may be probed using the procedure ofGrunstein and Hogness Proc. Natl. Acad. Sci. USA, 7:3961 (1975), orother available techniques as described in Sambrook et al. Briefly, inthis procedure, the DNA to be probed is immobilized on a membrane (e.g.,nitrocellulose or nylon filters) denatured, and prehybridized with abuffer containing 0-50% formamide, 0.75 M NaCl, 75 mM Na citrate, 0.02%(wt/v) each of bovine serum albumin, polyvinyl pyrollidone, and Ficoll,50 mM Na Phosphate (pH 6.5), 0.1% SDS, and 100 micrograms/ml carrierdenatured DNA. The percentage of formamide in the buffer, as well as thetime and temperature conditions of the prehybridization and subsequenthybridization steps depends on the stringency required. Oligomericprobes which require lower stringency conditions are generally used withlow percentages of formamide, lower temperatures, and/or longerhybridization times. Both non-radioactive and radioactive techniques canbe utilized. Probes containing more than 30 or 40 nucleotides such asthose derived from genomic sequences generally employ highertemperatures, e.g., about 40°-42° C., and a high percentage, e.g., 50%,formamide. Following prehybridization, 5'-³² P-labeled oligonucleotideprobe is added to the buffer, and the filters are incubated in thismixture under hybridization conditions. After washing, the treatedfilters are subjected to autoradiography or a non-radioactive techniquesuch as DIGOXIGENIN™ D/UTP labeling kit (Boehringer Mannheim,Indianapolis, Ind.), to show the location of the hybridized probe; DNAin corresponding locations on the original agar plates is used as thesource of the desired DNA.

The nucleotide sequence of an avian E. coli iss gene has about a 96.8%sequence identity in a 310 bp overlap, as shown in FIG. 1, when comparedto the entire sequence of a human E. coli iss gene (Chuba et al., Mol.Gen. Genet., 216:287-292 (1989)). Surprisingly, the avian E. coli issgene has an insertion at nucleotide 158 and a deletion at nucleotide189, that results in a frameshift in the resulting polypeptide sequence(SEQ ID NO.2). The 10 amino acid change located in the middle of theprotein creates an approximate 86.3% sequence identity in a 102 aminoacid overlap, as shown in FIG. 2, between the human ("Iss₋₋ Ec") andavian ("102 Iss") E. coli Iss polypeptides. The 102 Iss amino acidsequence and the Iss₋₋ Ec amino acid sequence are about 86.3% identicalin a 102 amino acid overlap. The low identity between these two E. coliIss sequences is due to a frameshift that has occurred in (Iss₋₋ Ec).The amino-terminal region of 102 Iss has structural featurescharacteristic of a cleavable signal sequence and spans amino acids 17to 22. Residues 17-21 of Iss are almost identical to residues 12-17 ofBor (FIG. 2).

When the avian E. coli iss sequence was examined with Gene Inspector™software (Textco, Inc., West Lebanon, N.H.), it was found that the avianE. coli Iss protein is predicted to have an isoelectric point ofapproximately 8.47, and at pH 7, is expected to have a net charge of+2.05. An Iss polypeptide is predicted to be approximately a 10-11 kDprotein containing 102 amino acids that is resistant to acid hydrolysis.Additionally, based on the Iss polypeptide's predicted foldingcharacteristics and hydropathy plots, Iss is likely to have a number ofaccessible sites, for example, sites not buried in the bacterialmembrane, that are antigenic.

The iss locus of the conjugative plasmid ColV, I-K94, is closely relatedto the phage lambda bor gene. Sequencing has shown that iss displays adiscrete 796 bp block of DNA homologous to a region from bp 46186 to46982 in the lambda sequence (Chuba et al., Mol. Gen. Genet.,216:287-292 (1989); Sanger et al., J, Mol. Biol., 162:729-773 (1982)).The overall identity between lambda bor and iss is about 81%, excludinggaps, as the avian E. coli iss nucleotide sequence has an approximate89.4% sequence identity in a 303 bp overlap with the lambda bor gene(Barondess et al., J. Bacteriol., 177:1247-1253 (1995); Barondess etal., Nature, 346:871-874 (1990)). The 102 Iss amino acid sequence andthe lamBor amino acid sequence are about 89.7% identical in a 97 aminoacid overlap.

C. Variants of the Nucleic Acid Molecules of the Invention

Nucleic acid molecules encoding amino acid sequence variants of an Isspolypeptide can be prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of an Iss polypeptide.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingamino acid substitution variants of an Iss polypeptide. This techniqueis well known in the art as described by Adelman et al., DNA 2:183(1983), and Zoller Nucleic Acids Res., 10:6487 (1982). Briefly, the DNAto be modified is packaged into phage as a single stranded sequence, andconverted to a double stranded DNA with DNA polymerase using, as aprimer, a synthetic oligonucleotide complementary to the portion of theDNA to be modified and having the desired modification included in itsown sequence. The resulting double-stranded DNA is transformed into aphage supporting host bacterium. As used herein, a "double-stranded DNAmolecule" refers to the polymeric form of deoxyribonucleotides (adenine,guanine, thymine or cytosine) in its normal, double-stranded helix. Thisterm refers only to the primary and secondary structure of the molecule,and does not limit it to any particular tertiary forms. Thus this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g., restriction fragments), viruses, plasmids and chromosomes. Indiscussing the structure of a particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5' to 3' direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). Cultures of the transformed bacteria, whichcontain replications of each strand of the phage, are plated in agar toobtain plaques. Theoretically, 50% of the new plaques contain phagehaving the mutated sequence, and the remaining 50% have the originalsequence. Replicates of the plaques are hybridized to a labeledsynthetic probe at temperatures and conditions that permit hybridizationwith the correct strand, but not with the unmodified sequence. Thesequences which have been identified by hybridization are recovered andthen cloned.

A DNA sequence encoding an Iss polypeptide can be synthetically preparedrather than isolated. The DNA sequence can be designed with theappropriate codons for an Iss amino acid sequence or variant thereof. Ingeneral, one will select preferred codons for the intended host if thesequence will be used for expression. The complete sequence is assembledfrom overlapping nucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge, Nature, 292:756(1981); Nambair et al., Science 2:1299 (1984); Jay et al., J. Biol.Chem. 2:6311 (1984). Additionally, synthetic iss oligonucleotidesequences may be prepared using an automated oligonucleotide synthesizeras described by Warner DNA, 3:401 (1984).

Additionally, nucleotide substitutions in SEQ ID NO:22 that can encode apolypeptide having SEQ ID NO:2 or variant thereof can be ascertained byreference to Table 1 and page D1 in Appendix D of Sambrook et al.,Molecular Cloning: A Laboratory Manual (1989), as well as Table 1hereinbelow. Nucleotide substitutions can be introduced into DNAsegments by methods well known to the art, some of which are describedabove. See, also, Sambrook et al., supra. Likewise, nucleic acidmolecules encoding other Iss polypeptides may be modified in a similarmanner. Thus, nucleic acid molecules encoding at least a portion of SEQID NO:2, or the complement thereto, may be modified so as to yieldnucleic acid molecules of the invention having "silent" nucleotidesubstitutions, or to yield nucleic acid molecules having nucleotidesubstitutions that result in amino acid substitutions (see polypeptideor peptide variants hereinbelow).

                  TABLE 1                                                         ______________________________________                                        Amino Acid   Codon                                                            ______________________________________                                        Phe          UUU, UUC                                                           Ser UCU, UCC, UCA, UCG, AGU, AGC                                              Tyr UAU,UAC                                                                   Cys UGU,UGC                                                                   Leu UUA, UUG, CUU, CUC, CUA, CUG                                              Trp UGG                                                                       Pro CCU, CCC, CCA, CCG                                                        His CAU, CAC                                                                  Arg CGU, CGC, CGA, CGG, AGA, AGG                                              Gln CAA, CAG                                                                  Ile AUU, AUC, AUA                                                             Thr ACU, ACC, ACA, ACG                                                        Asn AAU, AAC                                                                  Lys AAA, AAG                                                                  Met AUG                                                                       Val GUU, GUC, GUA, GUG                                                        Ala GCU, GCC, GCA, GCG                                                        Asp GAU, GAC                                                                  Gly GGU, GGC, GGA, GGG                                                        Glu GAA, GAG                                                                ______________________________________                                    

D. Preparation of Expression Cassettes and Vectors and TheirIntroduction into Host Cells

To prepare expression cassettes and vectors for transformation herein,the recombinant or preselected nucleic acid sequence or segment may becircular or linear, double-stranded or single-stranded. As used herein,a "vector," "expression vector," or "expression cassette" is a replicon,or a genetic element that functions as an autonomous unit of DNAreplication in vivo and capable of replication under its own control,such as a plasmid, a chromosome, a virus, phage or cosmid. Another DNAsegment may be attached to the replicon or genetic element so as tobring about the replication of the attached segment.

Expression cassettes or expression vectors for host cells ordinarilyinclude an origin of replication, a promoter located upstream from theIss coding sequence, together with a ribosome binding site, apolyadenylation site, and a transcriptional termination sequence. Thoseof ordinary skill will appreciate that some of the aforementionedsequences are not required for expression in certain hosts. For example,an expression vector for use with microbes need only contain an originof replication recognized by the host, a promoter that will finction inthe host and a selection gene.

An expression cassette is constructed according to the present inventionso that an avian E. coli iss coding sequence is located in the cassettewith the appropriate regulatory sequences, the positioning andorientation of the coding sequence with respect to the control sequencesbeing such that the coding sequence is transcribed under the "control"of the control sequences (e.g., RNA polymerase that binds to the DNAmolecule as the control sequences transcribes the coding sequence). Asused herein, a DNA "coding sequence" is that portion of a DNA sequence,the transcript of which is translated into a polypeptide in vivo whenplaced under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5' (amino terminus) and a translation stop codon at the 3' (carboxyterminus). A coding sequence can include, but is not limited to,prokaryotic sequences, genomic DNA sequences from eukaryotic DNA, cDNAfrom eukaryotic mRNA and even synthetic DNA sequences. A polyadenylationsignal and transcription termination sequence will usually be located 3'to the coding sequence.

The control sequences can be ligated to the coding sequence prior toinsertion into a cassette. As used herein, a coding sequence is "underthe control" of the promoter sequence in a cell when RNA polymerasewhich binds the promoter sequence transcribes the coding sequence intomRNA which is then in turn translated into the protein encoded by thecoding sequence. Alternatively, the coding sequence can be cloneddirectly into an expression cassette that already contains the controlsequences and an appropriate restriction site.

1. Transformations

Transformation may be by any known method for introducingpolynucleotides into a host cell, including, for example, packaging thepolynucleotide in a virus and transducing a host cell with the virus,and by direct uptake of the polynucleotide. The terms "transformed" or"transformation" or "stably transformed", as used herein, refer to theinsertion of an exogenous polynucleotide into a host cell, irrespectiveof the method used for the insertion, for example, direct uptake,transduction, f-mating or electroporation. The exogenous polynucleotidemay be maintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host genome. A cell has been"transformed" by exogenous DNA when such exogenous DNA has beenintroduced into the cell membrane. Exogenous DNA may or may not beintegrated (covalently linked) to chromosomal DNA making up the genomeof the cell. In prokaryotes and yeast, for example, the exogenous DNAmay be maintained on an episomal element such as a plasmid. With respectto eukaryotic cells, a stably transformed cell is one in which theexogenous DNA has become integrated into a chromosome so that it isinherited by daughter cells through chromosomal replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the exogenous DNA.

The transformation procedure used depends upon the host to betransformed. Bacterial transformation by direct uptake generally employstreatment with calcium or rubidium chloride (Cohen Proc. Natl. Acad.Sci. USA, 69:2110 (1972)). Yeast transformation by direct uptake may becarried out using the method of Hinnen et al. Proc. Natl. Acad. Sci.,75:1929 (1978). Mammalian transformations by direct uptake may beconducted using the calcium phosphate precipitation method of Graham andVan der Eb Virology, 52:546 (1978), or the various known modificationsthereof. Other methods for the introduction of recombinantpolynucleotides into cells, particularly into mammalian cells, that areknown in the art include dextran-mediated transfection, calciumphosphate mediated transfection, polybrene mediated transfection,protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of thepolynucleotides into nuclei.

As used herein, the term "recombinant nucleic acid," refers to a nucleicacid that has been derived or isolated from any appropriate source, thatmay be subsequently chemically altered in vitro, and later introducedinto target host cells. An example of recombinant DNA "derived" from asource, would be a DNA sequence that is identified as a useful fragmentencoding iss, or a fragment or a variant thereof, and which is thenchemically synthesized in essentially pure form. An example of such DNA"isolated" from a source would be a useful DNA sequence that is excisedor removed from said source by chemical means, for example, by the useof restriction endonucleases. The isolated sequence can then be furthermanipulated, such as, amplified for use in the invention, by themethodology of genetic engineering.

"Recombinant host cells," "host cells," "cells," "cell lines," cellcultures," and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellsthat can be, or have been used as recipients for a recombinant vector orother transfer DNA, and include the progeny of the original cell whichhas been transformed. It is understood that the progeny of a singleparental cell may not necessarily be completely identical in morphologyor in genomic or total DNA complement as the original parent, due tonatural, accidental or deliberate mutation.

2. Cellular hosts

Both prokaryotic and eukaryotic host cells may be used for expression ofdesired coding sequences. Appropriate control sequences that arecompatible with the designated host should be used. The term "controlsequences" is defined to mean DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Theterm "operably linked" is defined to mean that the nucleic acids areplaced in a functional relationship with another nucleic acid sequence.For example, DNA for a presequence or secretory leader is operablylinked to DNA for a polypeptide or fusion polypeptide if it is expressedas a pre-protein that participates in the secretion of the polypeptideor fusion polypeptide; a promoter or enhancer is "operably linked" to acoding sequence if it affects the transcription of the sequence if it ispositioned so as to facilitate translation. Generally, "operably linked"means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

The control sequences that are suitable for prokaryotic cells, forexample, include a promoter and optionally an operator sequence, and aribosome binding site. A "promoter" or "promoter sequence" is a DNAregulatory region to which RNA polymerase binds and initiatestranscription of a downstream (3' direction) coding sequence. Forpurposes of defining the present invention, the promoter sequence isbounded at its 3' terminus by the translation start codon of a codingsequence and extends upstream (5' direction) to include the minimumnumber of bases or elements necessary to initiate transcription atlevels detectable above background. Within the promoter sequence will befound a transcription initiation site, as well as protein bindingdomains (consensus sequences) responsible for the binding of RNApolymerase. Eukaryotic promoters will often, but not always, contain"TATA" boxes and "CAT" boxes. Prokaryotic promoters containShine-Dalgarno sequences in addition to the -10 and -35 consensussequences.

Eukaryotic cells are known to utilize promoters, polyadenylation signalsand enhancers. Expression control sequences for prokaryotes includepromoters, optionally containing operator portions and ribosome bindingsites. Among prokaryotic hosts, E. coli is most frequently used. Anumber of prokaryotic expression vectors are known in the art. See, forexample, U.S. Pat. Nos. 4,440,859; 4,436,815; 4,431,740; 4,431739;4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; seealso U.K. Pub. NOs. GB 2,121,054; GB 2,008,123; GB 2,007,675; andEuropean Pub. No. 103,395.

Transfer vectors compatible with prokaryotic hosts are commonly derivedfrom pBR322, a plasmid containing operons conferring ampicillin andtetracycline resistance, and various pUC vectors, which also containsequences conferring antibiotic resistance markers. These markers may beused to obtain successful transformants by selection. Preferably,expression vectors obtained commercially from Pharmacia are utilized andinclude pGEX-1λT (product No. 27-4805-01), pGEX-2T (product No.27-4801-01), pGEX-2TK (product No. 27-4587-01), pGEX-4T-1 (product No.27-4580-01), pGEX-4T-2 (product No. 27-4581-01), pGEX-4(product No.27-4583-01), pGEX-3X (product No. 27-4803-01), pGEX-5X-1 (product No.27-4584-01), pGEX-5X-2 (product No. 27-4585-01), pGEX-5X-3 (product No.27-4586-01), pGEX-6P-1 (product No. 27-4597-14 01), pGEX-6P-2 (productNo. 27-4598-01) and pGEX-6P-3 (product No. 27-4599-01). Expression ofiss in these vectors yields an GST-Iss fuision protein that can beemployed to prepare antibodies, immunogenic compositions, vaccines anddiagnostic kits. Alternatively, the Iss polypeptide can be cleaved fromGST with PreScission™ protease (Pharmacia Biotech. Inc.) or othersuitable enzyme, purified, and the Iss polypeptide can independently beemployed to prepare antibodies, immunogenic compositions, vaccines anddiagnostic kits.

Commonly used prokaryotic control sequences include the Beta-lactamase(penicillinase) and lactose promoter systems (Chang et al. Nature, 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al.Nucleic Acids Res., 8:4057 (1980)) and the lambda-derived PL promoterand N gene ribosome binding site (Shimatake et al. Nature, 292:128(1981)) and the hybrid tac promoter (De Boer et al. Proc. Natl. Acad.Sci. USA, 292:128 (1983)) derived from sequences of the trp and lac UV5promoters. The foregoing systems are particularly compatible with E.coli. If desired, other prokaryotic hosts such as strains of Bacillus orPseudomonas may be used with appropriate control sequences. Although thepromoters cited above are commonly used, other microbial promoters knowin the art, are also suitable.

For routine vector constructions, ligation mixtures are transformed intoE. coli strain HB101 or other suitable host, and successfultransformants selected by antibiotic resistance or other markers.Plasmids from the transformants are then prepared according to themethod of Clewell et al. Proc. Natl. Acad. Sci. USA, 62:1159 (1969),usually following chloramphenicol amplification (Clewell J, Bacteriol.,110:667 (1972)). The DNA is isolated and analyzed, usually byrestriction enzyme analysis and/or sequencing. Sequencing may be by thedideoxy method of Sanger et al. Proc. Natl. Acad. Sci. USA, 74:5463(1977) as further described by Messing et al. Nucleic Acids Res., 9:309(1981), or by the method of Maxam et al. Methods in Enzymology, 65:499(1980). Problems with band compression, which are sometimes observed inGC rich regions, are overcome by use of 7-deazoguanosine according toBarr et al. Biotechniques, 4:428 (1986).

Eukaryotic hosts include yeast and mammalian cells in culture systems.Saccharomyces cervisiae and Saccharomyces carlsbergensis are the mostcommonly used yeast hosts and are convenient fingal hosts. Yeastcompatible vectors carry markers which permit selection of successfultransformants by conferring prototrophy to auxotrophic mutants orresistance to heavy metals on wild-type strains. Yeast compatiblevectors may employ the 2 micron origin of replication (Broach et al.Meth. Enz., 101:307 (1983)), the combination of CEN3 and ARS1 or othermeans for assuring replication, such as sequences which will result inincorporation of an appropriate fragment into the host cell genome.

Control sequences for yeast vectors are known in the art and includepromoters for the synthesis of glycolytic enzymes (Hess et al. J. Adv.Enzyme Reg, 7:149 (1968)); (Holland et al. Biochemistry, 17:4900(1978)), including the promoter for 3 phosphoglycerate kinase (Hitzemanet al. J. Biol. Chem., 255:2073 (1980)). Terminators may also beincluded, such as those derived from the enolase gene (Holland et al. J.Biol Chem., 2:1385 (1981)). Particularly useful control systems arethose that comprise the glyceraldehyde-3 phosphate dehydrogenase (GAPDH)promoter or alcohol dehydrogenase (ADH) regulatable promoter,terminators also derived from GAPDH, and if secretion is desired, leadersequence from yeast alpha factor.

In addition, an operably linked transcriptional regulatory region andtranscriptional initiation region do not have to be ones that arenaturally associated in a wild-type organism. These systems aredescribed in detail in EPO 120,551, granted Aug. 1, 1990; EPO 116,201,granted Apr. 22, 1992; and EPO 164,556, granted Mar. 2, 1992, and arehereby incorporated herein by reference. Other yeast promoters, whichhave the additional advantage of transcription controlled by growthconditions, are the promoter regions for alcohol dehydrogenase 1 or 2,isocytochrome C, acid phosphatase, as well as enzymes responsible formaltose and galactose utilization.

Mammalian cell lines available as hosts for expression are known in theart. Suitable host cells for expressing Iss in higher eukaryotes includethe following: monkey kidney CVI line transformed by SV40 (COS-7, ATCCCRL 1651); baby hamster kidney cells (BHK, ATCC CRL 1651); Chinesehamster ovary-cells-DHFR (described by Urlaub and Chasin, PNAS, 77:4216(1980, USA)); mouse sertoli cells (TM4. Mather, J. P., Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70): African greenmonkey kidney cells (VERO-76, ATCC CRL-1587): human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A ATCC CRL 1442); human lung cells (W138,ATCC CCL 75): human liver cells (Hep G2 HB 8065); mouse mammary tumor(MMT 060652, ATCC CCL 51); rat hepatoma cells (HTC. M1.54. Baumann etal., J. Cell Biol., 85:1-8 (1980) and TRI cells (Mather et al., AnnalsN.Y. Acad. Sci., 3:44-68 (1982)).

Suitable promoters for mammalian cells are also known in the art andinclude viral promoters such as that from Simian Virus 40 (SV40) (Fierset al. Nature, 273:113 (1978)), Rous sarcoma virus (RSV), adenovirus(ADV), and bovine papilloma virus (BPV). Mammalian cells may alsorequire terminator sequences and poly A addition sequences; enhancersequences that increase expression can also be included, and sequenceswhich cause amplification of the gene may also be desirable. Thesesequences are known in the art. It will be appreciated that whenexpressed in mammalian tissue, a recombinant Iss may have highermolecular weight due to glycosylation. It is therefore intended thatpartially or completely glycosylated forms of Iss having molecularweights greater than provided by the amino acid back-bone are within thescope of this invention.

Vaccinia virus may be used to express foreign DNA and may be used invaccine preparation. In this case the heterologous DNA is inserted intothe Vaccinia genome. Techniques for the insertion of foreign DNA intothe vaccinia virus genome are known in the art, and use, for example,homologous recombination. The insertion of the heterologous DNA isgenerally into a gene that is non-essential in nature, for example, thethymidine kinase gene (tk), which also provides a selectable marker.Plasmid vectors that greatly facilitate the construction of recombinantviruses have been described (see, for example, Mackett et al. J. Virol.,49:857 (1984), Chakrabarti et al. Mol. Cell Biol. 5:3403 (1985); Moss,Gene Transfer Vectors for Mammalian Cells, (Miller and Calos, eds., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.), p.10 (1987)).Expression of the Iss polypeptide then occurs in cells or animalsubjects, e.g., avians, that are immunized with the live recombinantvaccinia virus.

Other systems for expression of eukaryotic or viral genomes includeinsect cells and vectors suitable for use in these cells. These systemsare known in the art, and include, for example, insect expressiontransfer vectors derived from the baculovirus Autographa californicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Expression vectors derived from this system usuallyuse the strong viral polyhedrin gene promoter to drive expression ofheterologous genes. Currently the most commonly used transfer vector forintroducing foreign genes into AcNPV is pAc373. The vector pAc373 alsocontains the polyhedrin polyadenylation signal and theampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli. Many other vectors, known to those of skillin the art, have also been designed for improved expression. Theseinclude, for example, pVL985 (which alters the polyhedrin start codonfrom ATG to ATT, and which introduces a BamHI cloning site 32 base pairsdownstream from the ATT; See Luckow and Summers Virology, 17:31 (1989)).Good expression of nonfused foreign proteins usually requires foreigngenes that ideally have a short leader sequence containing suitabletranslation initiation signals preceding an ATG start signal.

Methods for the introduction of heterologous DNA into the desired sitein the baculovirus virus are known in the art. (See Summer and Smith,Texas Agricultural Experiment Station Bulletin No. 1555; Smith et al.Mol. & Cell Biol., 3:2156-2165 (1983); and Luckow and Summers, supra(1989)). For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous recombination. Insertion can also be intoa restriction enzyme site engineered into the desired baculovirus gene.The inserted sequences may be those which encode all or varying segmentsof the polyprotein, or other open reading frames ("ORFs") which encodeviral polypeptides.

The signals for posttranslational modifications, such as signal peptidecleavage, proteolytic cleavage and phosphorylation, appear to berecognized by insect cells. The signals required for secretion andnuclear accumulation also appear to be conserved between theinvertebrate cells and vertebrate cells. Examples of the signalsequences from vertebrate cells which are effective in invertebratecells are known in the art, for example, the human interleukin 2 signal(IL2_(s)) which is a signal for transport out of the cell, is recognizedand properly removed in insect cells.

In a preferred embodiment, the product upon expression of an avian E.coli Iss polypeptide yields an Iss-fusion protein that can be purifiedby affinity chromatography using a commercially available kit with anaffinity for the fusion protein (Pharmacia, GST Gene Fusion System,Third Edition, Revision 1 (1997) (product No.18-1123-20), Bulk GSTPurification Module (product No. 27-4570-01), Redipack GST PurificationModule (product No. 27-4570-02). Once the Iss-fusion protein has beenpurified, Iss is cleaved from the fusion protein and utilized toimmunize mice, e.g., Balb/c. However, an Iss fusion protein can besubstituted as the immunogen if desired, although use of Iss alone ispreferred. Spleen cells from the immunized mice showing a response toIss or an Iss fusion protein are fused with myeloma cells to createhybridomas, and hybridomas producing antibody specific to Iss arepropagated, characterized and retained for use in immunologic assays todefine the association of Iss with avian E. coli virulence.

E. Polypeptides. Peptides and Variants Thereof

A recombinant or derived Iss polypeptide, as described above, is notnecessarily translated from a designated nucleic acid sequence, forexample, SEQ ID NO.22. Iss polypeptides, antigenic variants and subunitsthereof, or other Iss subunit polypeptides can be synthesized by thesolid phase peptide synthesis (or Merrifield) method or be prepared by avariety of methods known in the art. These and other methods of peptidesynthesis are also exemplified by U.S. Pat. Nos. 3,862,925; 3,842,067;3,972,859; 4,105,602 and 4,757,048.

The Merrifield method is an established and widely used method. It isdescribed in the following references: Stewart et al., Solid PhasePeptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield,J. Am. Chem. Soc., 85:2149 (1963); Meinenhofer in Hormonal Proteins andPeptides, Vol. 2, C. H. Li, ed., (Academic Press, 1973), pp. 48-267; andBarany and Merrifield in "The Peptides," Vol. 2, E. Gross and F.Meinenhofer, eds., Academic Press (1980), pp. 3-285.

The Merrifield synthesis method commences from the carboxy-terminal endof the peptide using an alpha-amino protected amino acid.Fluorenylmethyloxy-carbonyl (Fmoc) or t-butyloxycarbonyl (Boc)protective groups can be used for all amino groups even though otherprotective groups are suitable, and the first protected amino acids canbe esterified to chloromethylated polystyrene resin supports. Thepolystyrene resin support is preferably a copolymer of styrene withabout 0.5 to 2% divinyl benzene as a cross-linking agent which causesthe polystyrene polymer to be insoluble in certain organic solvents. SeeCarpino et al., J. Org. Chem., 37:3404 (1972); Meinenhofer, Int. J.Peat. Pro. Res., 11:246 (1978); and Merrifield, J. Am. Chem. Soc.,85:2149 (1963). The immobilized peptide is then N-deprotected and otheramino acids having protected amino groups are added in a stepwise mannerto the immobilized peptide. At the end of the procedure, the finalpeptide is cleaved from the resin, and any remaining protecting groupsare removed by treatment under acidic conditions, for example, with amixture of hydrobromic acid and trifluoroacetic acid. Alternatively, thecleavage from the resin may be effected under basic conditions, forexample, with triethylamine, where the protecting groups are thenremoved under acidic conditions. The cleaved peptide is isolated andpurified by means well known in the art, for example, by lyophilizationfollowed by either exclusion or partition chromatography onpolysaccharide gel media such as Sephadex G-25, or countercurrentdistribution. The composition of the final polypeptide may be confirmedby amino acid analysis after degradation of the polypeptide by standardmeans.

The synthesis may use manual techniques or be completely automated. Forexample, an Applied BioSystems 431A Peptide Synthesizer (Foster City,Calif.) or a Biosearch SAM II automatic peptide synthesizer (Biosearch,Inc., San Rafael, Calif.) can be employed following the directionsprovided in the instruction manual and reagents supplied by themanufacturer. Disulfide bonds between Cys residues can be introduced bymild oxidation of the linear peptide by KCN as taught in U.S. Pat. No.4,757,048 at column 20.

Salts of carboxyl groups of a polypeptide, peptide or variant of theinvention may be prepared in the usual manner by contacting the peptidewith one or more equivalents of a desired base, for example, a metallichydroxide base (e.g., sodium hydroxide), a metal carbonate orbicarbonate base, for example, sodium carbonate or sodium bicarbonate,or an amine base, for example, triethylamine or triethanolamine. Acidaddition salts of the peptide may be prepared by contacting the peptidewith one or more equivalents of the desired inorganic or organic acid,for example, hydrochloric acid.

Esters of carboxyl groups of the polypeptides may be prepared by any ofthe usual methods known in the art for converting a carboxylic acid orprecursor to an ester. One preferred method for preparing esters of thepresent polypeptides, when using the Merrifield synthesis techniquedescribed above, is to cleave the completed polypeptide from the resinin the presence of the desired alcohol either under basic or acidicconditions, depending upon the resin. Thus, the C-terminal of thepeptide when freed from the resin is directly esterified withoutisolation of the free acid.

Amides of the polypeptides of the present invention may also be preparedby techniques well known in the art for converting a carboxylic acidgroup or precursor, to an amide. A preferred method for amide formationat the C-terminal carboxyl group is to cleave the polypeptide from asolid support with an appropriate amine, or to cleave in the presence ofan alcohol, yielding an ester. This cleavage is followed by aminolysiswith the desired amine.

N-acyl derivatives of an amino group of a polypeptide, peptide orvariant of the invention may be prepared by utilizing an N-acylprotected amino acid for the final condensation, or by acylating aprotected or unprotected peptide. O-acyl derivatives may be prepared,for example, by acylation of a free hydroxy peptide or peptide resin.Either acylation may be carried out using standard acylating reagentssuch as acyl halides, anhydrides, acyl imidazoles, and the like. Both N-and O-acylation may be carried out together, if desired.Formyl-methionine, pyroglutamine and trimethyl-alanine may also besubstituted at the N-terminal residue of the polypeptide, peptide orvariant. Other amino-terminal modifications include aminooxypentanemodifications (see Simmons et al., Science 276:276 (1997)).

In addition, the amino acid sequence of an Iss polypeptide can bemodified so as to result in a specific polypeptide variant. Themodification includes the substitution of at least one amino acidresidue in the peptide for another amino acid residue, and includessubstitutions that utilize the D rather than L form, as well as otherwell known amino acid analogs.

One or more residues of the polypeptide or peptide can be altered, solong as the resultant variant is biologically active. Conservative aminoacid substitutions are preferred. For example, aspartic-glutamic asacidic amino acids; lysine/arginine/histidine as basic amino acids;leucine/isoleucine, methionine/valine, alanine/valine as hydrophobicamino acids; serine/glycine/alanine/threonine as hydrophilic aminoacids.

Conservative substitutions within the scope of the invention includethose shown in Table 2 under the heading of exemplary substitutions.More preferred substitutions are under the heading of preferredsubstitutions. After the substitutions are introduced, the resultingvariants are screened for biological activity.

                  TABLE 2                                                         ______________________________________                                        Original    Exemplary        Preferred                                          Residue Substitutions Substitutions                                         ______________________________________                                        Ala (A)     val; leu; ile    val                                                Arg (R) lys; gln; asn lys                                                     Asn (N) gln; his; lys; arg gln                                                Asp (D) glu glu                                                               Cys (C) ser ser                                                               Gln (Q) asn asn                                                               Glu (E) asp asp                                                               Gly (G) pro pro                                                               His (H) asn; gln; lys; arg arg                                                Ile (I) leu; val; met; ala; phe leu                                            norleucine                                                                   Leu (L) norleucine; ile; val; met; ile                                         ala; phe                                                                     Lys (K) arg; gln; asn arg                                                     Met (M) leu; phe; ile leu                                                     Phe (F) leu; val; ile; ala leu                                                Pro (P) gly gly                                                               Ser (S) thr thr                                                               Thr (T) ser ser                                                               Trp (W) tyr tyr                                                               Tyr (Y) trp; phe; thr; ser phe                                                Val (V) ile; leu; met; phe; ala; leu                                           norleucine                                                                 ______________________________________                                    

Amino acid substitutions falling within the scope of the invention, are,in general, accomplished by selecting substitutions that do not differsignificantly in their effect on maintaining (a) the structure of thepeptide backbone in the area of the substitution, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain. Naturally occurring residues are divided into groupsbased on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gIn, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic; trp, tyr, phe.

The invention also envisions polypeptide or peptide variants withnon-conservative substitutions. Non-conservative substitutions entailexchanging a member of one of the classes described above for another.

Acid addition salts of the polypeptide, peptide or variant thereof or ofamino residues of the polypeptide, peptide or variant may be prepared bycontacting the polypeptide, peptide, variant or amine thereof with oneor more equivalents of the desired inorganic or organic acid, such as,for example, hydrochloric acid. Esters of carboxyl groups of thepolypeptides or peptides may also be prepared by any of the usualmethods known in the art.

F. Preparaion of Iss Antibodies

The immunogenic Iss polypeptides or variants thereof prepared asdescribed above are used to produce antibodies, including polyclonal andmonoclonal. As used herein, the term "antibody" refers to a polypeptideor group of polypeptides which are comprised of at least one antibodycombining site. An "antibody combining site" or "binding domain" isformed from the folding of variable domains of an antibody molecule(s)to form three-dimensional binding spaces with an internal surface shapeand charge distribution complementary to the features of an epitope ofan antigen, which allows an immunological reaction with the antigen. Anantibody combining site may be formed from a heavy and/or a light chaindomain (VH and VL, respectively), which form hyper-variable loops whichcontribute to antigen binding. The term antibody also includes, forexample, vertebrate antibodies, hybrid antibodies, chimeric antibodies,altered antibodies, univalent antibodies, monoclonal and polyclonalantibodies, the Fab proteins and single domain antibodies.

If polyclonal antibodies are desired, a selected animal (e.g., mouse,rabbit, goat, horse or bird) is immunized with an immunogenic Isspolypeptide or Iss-fusion protein. Serum from the immunized animal iscollected and treated according to known procedures. If serum containingpolyclonal antibodies to an Iss antigen contains antibodies to otherantigens, the polyclonal antibodies can be purified by immuno-affinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art, see for example, Mayer and Walker eds.Immunochemical Methods in Cell and Molecular Biology (Academic Press,London) (1987).

Monoclonal antibodies directed against Iss antigens or polypeptides canbe readily produced by one skilled in the art. The general methodologyfor making monoclonal antibodies by hybridomas is well known. Immortalantibody-producing cell lines can be created by cell fuision, and alsoby other techniques such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus. See, e.g.,Schreier et al. Hybridoma Techniques, (1980); Hammerling et al.Monoclonal Antibodies and T-cell Hybridomas, (1981); Kennett et al.Monoclonal Antibodies (1980); see also, U. S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500; 4,491,632; and4,493,890. Panels of monoclonal antibodies produced against Iss antigenscan be screened for various properties, for example, isotype, epitopeaffinity, etc.

Preferably, an Iss polypeptide, peptide, variant or subunit thereof, orIss-fusion protein, is utilized to immunize mice for the preparation ofmonoclonal antibodies. If it is determined that an Iss polypeptide, afragment or variant thereof does not elicit a strong humoral response inthe immunized mice, a larger and possibly more immunogenic fusionprotein (e.g., GST-Iss) can be used as an immunogen. However, regardlessof the immunogen used, antibody specificity is determined using Issalone. Briefly, antigen (an Iss polypeptide, variant or a fragmentthereof, or GST-Iss) is emulsified in Complete Freund's Adjuvant and theemulsion used to immunize Balb/c mice (about 50-100 μg antigen per mousegiven IP). Mice are boosted with an emulsion of antigen-IncompleteFreund's Adjuvant twice at about 10 day intervals (about 50-100 μgantigen each, given IP). About ten days after the second booster, anantigen-capture ELISA is run to determine the response of the mice toIss.

The detection of antibody responses specific for the polypeptide can beused in ELISA-based immunoassays for the serodiagnosis of a septicemicdisease or virulent, complement resistance E. coli. The ELISA isperformed by using Iss to coat wells of microtiter plates. Afterovernight incubation, coated plates are washed thoroughly, andnonspecific binding sites are blocked. After incubation, plates arethoroughly washed. The primary antibody, i.e. antibody contained in thesera from mice immunized with Iss or GST-Iss, is diluted and added tothe microtiter plate wells. Following additional washes, a goatanti-mouse IgG- and IgM- alkaline phosphatase conjugate is added to thewells. After incubation and thorough washing, the substrate for thephosphatase, p-nitrophenyl phosphate, is added to the wells. Plates areincubated in the dark for about 10-45 minutes. Subsequently, changes inabsorbance of the plate's contents are read at 405 nm with a microplatespectrophotometer as an indication of mouse response to Iss antigen.With the identification of a positive antibody, production ofmonoclonals can proceed. If a positive antibody is not identified, moreboosters may be used, or techniques to increase the immunogenicity ofIss can be implemented as stated above. Alternatively, GST-Iss can beused as the immunogen.

Responding mice are given a final booster consisting of about 5-100 μg,preferably 25-50 μg of antigen, preferably without adjuvant,administered intravenously. Three to five days after final boosting,spleens and sera are harvested from all responding mice, and sera isretained for use in later screening procedures. Spleen cells areharvested by perfusion of the spleen with a syringe. Spleen cells arecollected, washed, counted and the viability determined via a viabilityassay. Spleen and SP2/0 myeloma cells (ATCC, Rockville, Md.) that havebeen screened for HAT sensitivity and absence of bacterial contaminationare combined, the suspension pelleted by centrifugation, and the cellsfused using polyethylene glycol solution. The "fused" cells areresuspended in HT medium (RPMI supplemented with 20% fetal bovine serum(FBS), 100 units of penicillin per ml, 0.1 mg of streptomycin per ml,100 μM hypoxanthine, 16 μM thymidine, 50 μM 2-mercaptoethanol and 30%myeloma-conditioned medium) and distributed into the wells of microtiterplates. Following overnight incubation at 37° C. in 5% CO₂, HATselection medium (HT plus 4 μM aminopterin) is added to each well andthe cells fed according to accepted procedures known in the art. Inapproximately 10 days, medium from wells containing visible cell growthare screened for specific antibody production by ELISA. Only wellscontaining hybridomas making antibody with specificity to Iss, orGST-Iss, are retained. The ELISA is performed as described above, exceptthat the primary antibody added is contained in the hybridomasupernatants. Appropriate controls are included in each step.

This process generates several hybridomas producing monoclonalantibodies to Iss polypeptides, or an Iss-ftision protein, e.g.,GST-Iss. Hybridoma cells from wells testing positive for anti-Iss, oranti-GST-Iss antibodies are cloned by limiting dilution and re-screenedfor anti-Iss antibody production using ELISA. Cells from positive wellsare subcloned to ensure their monoclonal nature. The most reactive linesare then expanded in cell culture and samples are frozen in 90% FBS-10%dimethylsulfoxide. All monoclonal antibodies are characterized using acommercial isotyping kit (BioRad Isotyping Panel, Oakland, Calif.) andpartially purified with ammonium sulfate precipitation followed bydialysis. Further purification is performed using protein-A affinitychromatography.

Antibodies, both monoclonal and polyclonal, that are directed againstIss antigens are particularly useful in diagnosis. Those that areneutralizing are useful in passive immunotherapy and treatment.Monoclonal antibodies, in particular, may be used to raise anti-idiotypeantibodies.

The term "treatment," as used herein, refers to prophylaxis and/ortherapy of an avian subject diagnosed with, exhibiting characteristicsor symptoms of, various E. coli infections including, but not limitedto, colibacillosis, coligranuloma, peritonitis, salpingitis, synovitis,omphalitis and air sacculitis. The term "therapy" refers to providingtherapeutic benefit or effect to an avian subject such that the subjectexhibits few or no symptoms of a septicemic disease or other relateddiseases. Such treatment can be accomplished by administration ofnucleic acids, polypeptides or antibodies of the instant invention.

G. Preparation of Immunogenic Compositions and Vaccines of the Invention

The preparation of immunogenic compositions or vaccines that containimmunogenic polypeptide(s), peptides, and polypeptides encoded by thenucleic acid molecules of the invention as an active ingredient areknown to one skilled in the art. As used herein, the term "immunogeniccomposition" refers to a composition or preparation administered in anamount effective to raise antibodies in a recipient and further providessome therapeutic benefit or effect so as to result in an immune responsethat inhibits or prevents a septicemic disease in avian, or so as toresult in the production of antibodies to a virulent complementresistant avian E. coli isolate, or polypeptide or peptide employed asan immunogen. Both local and systemic administration is contemplated.Systemic administration is preferred.

The term "vaccine" refers to the process of immunization and theadministration of an antigen or a suspension of antigens, derived fromeither bacteria or viruses, that upon administration, will produceactive immunity and provide protection against those viruses or bacteriaor related viruses or bacteria, utilizing either conventional,traditional or recombinant techniques, e.g. those involving recombinantDNA technology or synthetic peptides. Typically, such immunogeniccompositions and vaccines are prepared as injectables, either as liquidsolutions or suspensions. Solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thepreparation may also be emulsified, or the polypeptide encapsulated inliposomes.

The active immunogenic ingredients are often mixed with excipients ordiluents that are pharmaceutically acceptable as carriers and compatiblewith the active ingredient. The term "pharmaceutically acceptablecarrier" refers to a carrier(s) that is "acceptable" in the sense ofbeing compatible with the other ingredients of a composition and notdeleterious to the recipient thereof. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the immunogeniccomposition or vaccine may contain minor amounts of auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents, and/oradjuvants which enhance the effectiveness of the immunogenic compositionor vaccine.

Examples of adjuvants or carriers that may be effective include but arenot limited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalnitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL +TDM +CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against an immunogenicpolypeptide containing an Iss antigenic sequence resulting fromadministration of the polypeptide in immunogenic compositions orvaccines that are also comprised of the various adjuvants.

H. Dosages, Formulations and Routes of Administration of Nucleic AcidMolecules and Polypeptides of the Invention

The nucleic acid molecules, polypeptides or peptides of the inventionare preferably administered to an avian subject so as to result in animmune response specific for the polypeptide, including the polypeptideencoded by the nucleic acid molecules of the invention or peptide. Theimmunogenic compositions and vaccines of the present invention areconventionally administered parenterally, by injection, for example,either subcutaneously or intramuscularly either as liquid solutions orsuspensions. Solid forms suitable for suspension in a liquid vehicleprior to injection or infusion can also be prepared. Additionalformulations that are suitable for administration include oralformulations. Oral formulations include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate and the like. Iss polypeptides may be formulated into animmunogenic composition or vaccine as neutral or salt forms. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain about10%-95% of active ingredient, preferably about 25%-70%.

Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids such as acetic, oxalic, tartaric, maleic, and thelike. Salts formed with the free carboxyl groups may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides and such organic bases as isopropylamine,trimethylamine, ethylamino ethanol, histidine, procaine, and the like.

Immunogenic compositions and vaccines comprising nucleic acid molecules,polypeptides or peptides of the instant invention, are administered toan animal, e.g., chicken, turkey, and other avian subjects, in a mannercompatible with the dosage formulation, and in such amount as will beprophylactically and/or therapeutically effective and result in animmune response that is specific for an Iss polypeptide, Iss peptide, orIss polypeptide encoded by a nucleic acid molecule. It is very common toexpress dosage units in mg/kg (i.e., mg/kg of body weight) or, if acontinuing series of doses over many days is contemplated, mg/kg/day. Animmunogenic composition or vaccine of the invention will usually containan effective amount, e.g., an amount capable of eliciting an immuneresponse in an avian subject, of an Iss polypeptide in conjunction witha conventional, pharmaceutically acceptable carrier. The dosage willvary depending upon the specific purpose for which the protein is beingadministered. The prepared compounds and compositions can beadministered to avian subjects for veterinary use, such as for use withdomestic or farm animals.

In general, the dosage required for efficacy will range from about 0.003to 100 mg/kg, preferably about 0.05 to 50 mg/kg, and more preferably 0.5to 30 mg/kg, although other dosages can provide beneficial effects. Adosing method as described in Borch et al. U.S. Pat. No. 5,035,878,provides additional guidance. Although Borch et al. is directed tomammals, the weight of an avian subject can readily be substituted.Dosage, however, may depend on the avian subject to be treated, capacityof the avian subject's immune system to synthesize antibodies, and thedegree of protection desired. Precise amounts of active ingredientrequired to be administered may depend on the judgment of thepractitioner and may be peculiar to the avian subject to be immunized.

The immunogenic composition or vaccine can be given in a single doseschedule, or preferably in a multiple dose schedule. A multiple doseschedule is one in which a primary course of vaccination may be withapproximately 1-10 separate doses, followed by other doses given atsubsequent time intervals required to maintain and or reenforce theimmune response, for example, at about 1-4 months for a second dose, andif needed, a subsequent dose(s) after several months. The dosage regimenwill also, at least in part, be determined by the need of the aviansubject and be dependent upon the judgment of the practitioner. Inaddition, the immunogenic composition or vaccine containing animmunogenic Iss antigen(s), Iss polypeptide or Iss fusion polypeptidecan be administered in conjunction with other immunoregulatory agents,for example, immune globulins.

Administration of sense or antisense nucleic acid molecules can beaccomplished through the introduction of cells transformed with anexpression vector, as described above, comprising the nucleic acidmolecule (see for example, WO 93/02556) or the administration of thenucleic acid molecule (see, for example, Felgner et al., U.S. Pat. No.5,580, 859, Pardoll et al., Immunity 3:165 (1995); Stevenson et al.,Immunol. Rev. 145:211 (1995); Molling, J. Mol. Med. 75:242 (1997);Donnelly et al., Ann. N.Y. Acad. Sci. 772:40 (1995); Yang et al., Mol.Med. Today 2:476 (1996); Abdallah et al., Biol. Cell 85:1 (1995)).Pharmaceutical formulations, dosages and routes of administration fornucleic acids are generally disclosed, for example, in Felgner et al.,and are fully applicable to avian subjects.

I. Immunoassay and Diagnostic Kits

Polypeptides that react immunologically with serum containing Issantibodies or Iss-fusion protein antibodies are useful in immunoassaysto detect presence of avian E. coli Iss in biological samples. Examplesof useful antibodies include those derived from, expressed from, orencoded within the Iss clones described in Example 1, and compositesthereof, and the antibodies raised against the Iss antigens in thesepolypeptides.

Design of an immunoassay is subject to a great deal of variation, andmany formats are known in the art. Ideally the immunoassay will utilizeat least one antigen derived from an avian E. coli Iss. In oneembodiment, the immunoassay can utilize a combination of antigensderived from Iss or an Iss fusion polypeptide. These antigens can bederived from the same or from different Iss polypeptides, and may be inseparate recombinant or natural polypeptides, or together in the samerecombinant polypeptides.

An immunoassay may use, for example, a monoclonal antibody directedtowards an Iss antigen(s) or Iss fusion polypeptide, a combination ofmonoclonal antibodies directed towards several Iss antigens or Issfusion polypeptides, polyclonal antibodies directed towards the same Issantigen or Iss fusion polypeptide, or polyclonal antibodies directedtowards different Iss antigens or Iss fusion polypeptides. Protocols maybe based, for example, upon competition, or direct reaction, or sandwichtype assays. Protocols may also, for example, use solid supports, or maybe by immunoprecipitation. Most assays involve the use of labeledantibody or polypeptide. The labels may be, for example, enzymatic,fluorescent, chemiluminescent, radioactive, or dye molecules. Assayswhich amplify the signals from the probe are also known. Examples ofthese are assays that utilize biotin and avidin, and enzyme-labeled andmediated immunoassays, such as ELISA assays.

Typically, an immunoassay for an anti-Iss antibody(s) will involveselecting and preparing the test sample suspected of containing theantibodies, such as a biological sample, then incubating it with anantigenic (i.e., epitope-containing) Iss polypeptide(s) or Iss fusionpolypeptide(s) under conditions that allow antigen-antibody complexes toform, and then detecting the formation of such complexes. Suitableincubation conditions are well known in the art. The immunoassay may be,without limitation, in a heterogeneous or in a homogeneous format and ofa standard or competitive type.

In a heterogeneous format, the polypeptide is typically bound to a solidsupport to facilitate separation of the sample from the polypeptideafter incubation. Examples of solid supports that can be used arenitrocellulose (e.g., in membrane or microtiter well form), polyvinylchloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g.,in beads or microtiter plates, polyvinylindine fluoride (known aslmmulon™)), diazotized paper, nylon membranes, activated beads andProtein A beads. For example, Dynatech Immulon™ 1 or Imnulon™ 2microtiter plates or 0.25 inch polystyrene beads (Precision PlasticBall) can be used in the heterogeneous format. The solid supportcontaining the antigenic polypeptide is typically washed afterseparating it from the test sample, and prior to detection of boundantibodies. Both standard and competitive formats are known in the art.

In a homogeneous format, the test sample is incubated with antigen insolution. For example, it may be incubated under conditions that willprecipitate any antigen-antibody complexes that are formed. Bothstandard and competitive formats for these assays are known in the art.

In a standard format, the amount of Iss antibodies forming theantibody-antigen complex is directly monitored. This may be accomplishedby determining whether labeled anti-xenogenic antibodies that recognizean antigen on anti-Iss antibodies will bind due to complex formation. Ina competitive format, the amount of Iss antibodies in the sample isdeduced by monitoring the competitive effect on the binding of a knownamount of labeled antibody (or other competing ligand) in the complex.

Complexes formed comprising anti-Iss antibody (or, in the case ofcompetitive assays, the amount of competing antibody) are detected byany of a number of known techniques, depending on the format. Forexample, unlabeled Iss antibodies in the complex may be detected using aconjugate of anti-xenogenic Ig complexed with a label, (e.g., an enzymelabel).

In immunoassays where Iss polypeptides are the analyte, the test sample,typically a biological sample, is incubated with anti-Iss antibodiesunder conditions that allow the formation of antigen-antibody complexes.Various formats can be employed. For example, a "sandwich assay" may beemployed, where antibody bound to a solid support is incubated with thetest sample; washed; incubated with a second, labeled antibody to theanalyte, and the support is washed again. Analyte is detected bydetermining if the second antibody is bound to the support. In acompetitive format, which can be either heterogeneous or homogeneous, atest sample is usually incubated with antibody and a labeled, competingantigen is also incubated, either sequentially or simultaneously. Theseand other formats are well known in the art.

Kits suitable for immunodiagnosis and containing the appropriate labeledreagents are constructed by packaging the appropriate materials. Thesekits include the polypeptides of the invention containing Iss antigens,or antibodies directed against Iss epitopes, in suitable containers,along with the remaining reagents and materials required for the conductof the assay packaged in preselected amounts, as well as a suitable setof assay directions. All of these items are contained within the outerpackaging of the kit, which may be a box, envelope, or the like. Theassay directions preferably comprises instruction means such as aprinted insert, a label, a tag, a cassette tape and the like,instructing the user in the practice of the assay format.

Additionally, iss probes can be packaged into diagnostic kits. Suchdiagnostic kits can include avian E. coli iss nucleic acid probe(s),that may be labeled. As used herein, the term "probe" refers to apolynucleotide that forms a hybrid structure with a sequence in a targetregion, due to complementarity of at least one sequence in the probewith a sequence in the target region. Specifically a probe is a nucleicacid sequence between 10 and 500 base pairs in length that containsspecific nucleotide sequences that specifically and preferentiallyhybridize under predetermined conditions to nucleic acid sequences of anavian E. coli iss nucleic acid sequence.

Alternatively, the probe DNA may be unlabeled and the ingredients forlabeling the probe may be included in separate containers in the kit.The kit may also contain other suitably packaged reagents and materialsneeded for the particular hybridization protocol (e.g., standards) aswell as instructions for conducting the test.

For example, one such diagnostic kit for detecting or determiningantibodies to Iss or an Iss fuision polypeptide comprises packagingcontaining, separately packaged: (a) a solid surface, such as a fibroustest strip, a multi-well microtiter plate, a test tube, or beads, havingbound thereto a polypeptide, peptide, variant, or subunit, of SEQ ID NO.2 and (b) labeled anti-avian immunoglobulin. A second embodiment of adiagnostic kit for detecting or determining Iss comprises packagingcontaining, separately packaged: (a) a solid surface having boundthereto antibodies to the polypeptide of SEQ ID NO. 2 or a fragmentthereof; and (b) a known amount of (i) antibodies specific to Iss or anIss fusion polypeptide or (ii) antibodies to Iss that comprise adetectable label, or a binding site for a detectable label. A thirdembodiment of a diagnostic kit for detecting Iss or an Iss fusionpolypeptide can comprise, in packaged association, separately packagedamounts of: (a) an Iss-specific antibody; (b) the labeled polypeptide ofSEQ ID NO. 2 or a fragment thereof, and (c) an anti-avianimmunoglobulin.

All publications, patents and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 Identification, cloning and sequence of an avian E. coli issgene

A wild-type E. coli isolate was obtained from the serum of a chick withsystemic colibacillosis. The isolate was identified as an O2 serotype.The virulence of the isolate was determined by embryo lethality assay.Twelve-day-old embryonated eggs were obtained from Seaboard, Athens,Georgia. Overnight cultures of the isolates that were tested, includingvirulent and avirulent control organisms, were pelleted bycentrifugation, washed twice in phosphate-buffered saline (1×PBS) andresuspended to a concentration of 10² colony-forming units per 0.10 mlof PBS. The final concentration was then confirmed by viable counts. The0.1-ml inoculum was then injected into the allantoic cavity of eachembryo. Twenty embryos per isolate were utilized. Inoculated embryoswere incubated at 37° C. and counted daily for 4 days to identify deadembryos (Minshew et al., Infect Immun. 20:50-54 (1978); Nolan et al.,Avian Dis. 36:395-397 (1992)).

Inoculation of the isolate into chick embryos resulted in death of 98%of the embryos, indicating that the isolate was virulent. The isolatealso was resistant to the lytic effects of complement, which did notdegrade C3, and limited C3 deposition on the cell surface of thebacterium when compared with a complement-sensitive mutant of thiswild-type isolate.

The isolate contained several plasmids including a 100-kb plasmid thatproduced Colicin V. The isolate also produced the siderophores,enterobactin and aerobactin. The isolate was non-hemolytic on blood agar(Nolan et al., Avian Dis., 36:398-402 (1992)), motile (Nolan et al.,Avian Dis., 36:395-397 (1992)), and lacked K1 antigen, capsule, and typeI pili. Additionally, the isolate contained the traT(Nolan et al., AvianDis., 3:146-150 (1994)) and iss genes, had a smooth LPS (Nolan et al.,Avian Dis., 38:146-150 (1994)), was resistant to rough-specificbacteriophages (Nolan et al., Avian Dis., 38:146-150 (1994)) and to theantibiotics, streptomycin, sulfisoxazole and tetracycline.

The complete iss gene from this isolate was obtained by PCRamplification utilizing the following oligonucleotide primer pair:

5'-GTGGCGAAAACTAGTAAAACAGC-3' (SEQ ID NO.3) and

5'-CGCCTCGGGGTGGATAA-3' (SEQ ID NO.4).

The primer pair was selected using DNASTAR's Primer Select Program(Madison, Wis.). The resultant 760 bp PCR product (SEQ ID NO.1) wassequenced and cloned into a pGEM-T vector (Promega Corp., Madison, Wis.)(Sambrook, Fitsch & Maniatis, Molecular Cloning; A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989); DNA Cloning, Volumes I andII (D. N. Glover ed. 1985)). The resulting iss plasmid clones were alsosequenced to confirm their identities using standard procedures with aLICORT automated sequencer (Lincoln, Nebr.). The iss DNA sequence wascompared with the iss sequence from an isolate of a human E. coli andthe sequence of lambda bor (Barondess et al., J. Bacteriol.,177:1247-1253 (1995); Barondess et al., Nature, 346:871-874 (1990);Chuba et al., Mol. Gen. Genet., 216:287-292 (1989)) using LasergeneSoftware (DNASTAR) and FASTA (EMBL, Heidelberg, Germany). The DNAsequence alignment of those three genes is shown in FIG. 1. The avian E.coli iss sequence was submitted to GENBANK on Jan. 10, 1998, and hasbeen assigned accession number AF042279.

EXAMPLE 2 Expression of Iss

Two amplified iss sequences, one containing a full-length iss sequenceas shown in FIG. 1 (SEQ ID NO.1), and the other containing a truncatediss sequence wherein nucleotides 1-72 (FIG. 1) were deleted, were clonedin frame into the expression vector, pGEX-6P-3 (Pharmacia Biotech Inc.,Piscataway, N.J.), generating plasmid clones pLN321 and pLN322respectively. The pGEX-6P-3 vector was used in combination with a GSTGene Fusion System Purification Kit (Pharmacia Biotech Inc., Piscataway,N.J.) and provided a complete system for expressing and purifyingresulting Iss polypeptides.

Once cloned into the pGEX-6P-3 expression vector, both plasmid cloneswere sequenced and shown, by DNA sequencing, to be fused to the GSTvector in the proper reading frame (FIG. 3, showing pLN321 only). ThepLN321 construct was then used to transform a protease-deficient E. colistrain BL21 (Pharmacia Biotech Inc., Piscataway, N.J.). Proteinexpression was induced with IPTG (Pharmacia Biotech Inc., Piscataway,N.J.). Following induction, the E. coli were lysed and the lysates wereanalyzed by SDS-PAGE. The crude total protein preparations showed thatthe uninduced and induced E. coli containing these constructs differedin their expression of a 37-kD protein band (FIG. 4). The 37-kD band,lane 3I of FIG. 4, corresponds in size to the predicted GlutathioneS-Transferase-Iss (GST-Iss) fusion protein, as the Iss polypeptide has amolecular weight of approximately 10-11 kD, and GST has a molecularweight of approximately 26 kD. The 37-kD product obtained from theiss-pGEX-6P-3 construct, was found in the induced bacterial lysate, andnot in the uninduced lysate.

To further confirm the identity of the 37-kD band observed in theinduced bacterial lysate, the proteins of these lysates were transferredfrom the SDS-PAGE gel to a PVDF membrane (BioRad, Oakland, Calif.). ThePVDF membrane was then probed with a GST monoclonal antibody (PharmaciaBiotech Inc., Piscataway, N.J.)). After washing, bound antibody wasdetected using a secondary antibody conjugated to alkaline phosphatasein the presence of the color reagent, BCIP/NBT enzyme substrate (BioRad,Oakland, Calif.). The 37-kD band was recognized by anti-GST, therebyconfirming that the 37-kD band represented a GST-Iss fusion product(FIG. 5).

EXAMPLE 3 Purification and isolation of Iss

Polypeptides prepared from the pGEX-6P-3 expression vector yielded aglutathione S-transferase-Iss ("GST-Iss") fusion polypeptide productthat is readily purified from the bacterial lysates by affmitychromatography under mild, non-denaturing conditions. Specifically, abacterial sonicate is applied to a column of glutathione sepharose 4B at4° C. and washed three times with 10 bed volumes of 1×PBS. Glutathioneelution buffer (10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0)) isadded to the column and incubated at room temperature (about 22-25° C.)for 10 minutes, and the fusion protein is eluted. Eluates recovered fromthe column contain the fusion protein.

Alternatively, after expression of GST-Iss in E. coli, the bacteria arelysed by sonication, and the insoluble material is pelleted and removed,and the supernatant passed through a slurry of Glutathione Sepharose 4B(Pharmacia Biotech Inc., Piscataway, N.J.) to permit binding of theGST-Iss fusion polypeptide to the Sepharose beads. To remove GST-Issfrom other cellular proteins, the "bead-bound" fusion polypeptide ispelleted by centrifugation and washed with 1×PBS. The desired product iseluted from the Sepharose by the addition of reduced glutathione(Pharmacia Biotech Inc., Piscataway, N.J.). Upon removal from theSepharose beads, the purified GST-Iss fusion product is cleaved into Issand GST by a site-specific protease, such as PreScission Protease(Pharmacia Biotech Inc., Piscataway, N.J.), and the remaining GST isseparated from Iss by the same procedure used to purify the GST-Issfusion polypeptide. The resulting polypeptide products are then analyzedby SDS-PAGE.

EXAMPLE 4 Presence of iss in avian E. coli isolates

Two hundred and ten E. coli isolates from poultry clinically diagnosedwith colibacillosis were obtained from several locations in the UnitedStates. Fifty-six E. coli isolates from the feces of apparently healthypoultry were obtained from the University of Georgia or were frompoultry in North Dakota. The identity of all E. coli isolates wasconfirmed using API20E Strips (bioMerieux, Vitek, Inc., Hazelwood, Mo.).Non-E. coli strains (Table 3) used to evaluate the specificity of theprobes were obtained from the NDSU Veterinary Diagnostic Laboratory.

                  TABLE 3                                                         ______________________________________                                        Bacillus subtilis   Micrococcus luteus                                          Citrobacter freundii Proteus mirabilis                                        Enterobacter aerogenes Pseudomonasfluorescens                                 Enterobacter cloacae Rhodococcus erythropolis                                 Hafria alvei Staphylococcus aureus                                            Klebsiella oxytoca Staphylococcus epidermidis                                 Klebsiella pneumoniae Streptococcus faecalis                                  Salmonella typhimurium (Copenhagen)                                         ______________________________________                                    

All isolates were maintained in LB broth (Difco Laboratories, Detroit,Mich.) supplemented with 20% glycerol and stored at -70° C. prior touse. When subjected to total DNA isolation procedures, sample isolateswere grown in BHI (Difco Laboratories, Detroit, Mich.), with or withoutantibiotics, overnight at 37° C. In preparation for amplificationprocedures, isolates were grown on MacConkey agar (Difco Laboratories,Detroit, Mich.) overnight at 37° C. For colony blotting procedures, testorganisms were grown on LB agar (Difco Laboratories, Detroit, Mich.)overnight at 37° C.

Probe Construction

A cvaC probe was prepared by digesting plasmid pHK11 (Dr. R. E. Wooley,University of Georgia, Athens, Ga.) with the restriction enzymes EcoRIand BglII, which were obtained from Promega Corp. (Madison, Wis.) toyield a 1.9 kb fragment (Gilson et al., J. Bacteriol. 169:1466-2470(1987)). To obtain a traT probe (Moll et al., Infect. Immun., 2:359-367(1980); Montenegro et al., J. Gen. Microbiol., 131:1511-1521 (1985)),the plasrnid pKT107 (Dr. F. C. Cabello, New York Medical College,Valhalla, N.Y.) was digested with BstEII to yield a 700 bp fragment.

Gene probes for ompA and iss were generated through DNA amplificationtechniques. Primer sequences useful to amplify ompA and iss sequenceswere selected using Lasergene software (DNAStar, Inc., Madison, Wis.)based on published sequences for these genes (Beck et al., Nuc. AcidsRes., 8:3011-3024 (1980); Chuba et al., Mol. Gen. Genet., 216:287-292(1989)) and were obtained from Genosys Biotechnologies, Inc., TheWoodlands, Tex. Primers employed in the detection of ompA and iss areshown in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Name    Sequences (5' to 3')                                                                            SEQ ID NO.                                                                           Tm                                           __________________________________________________________________________    ompA                                                                            upper primer CTTGCGGAGGCTTGTCTGAG 9 54.9                                                                     ° C.                                    lower primer AGGCATTGCTGGGTAAGGAA 10 53.7° C.                          iss                                                                           upper primer GTGGCGAAAACTAGTAAAACAGC 3 52.1° C.                        lower primer CGCCTCGGGGTGGATAA 4 53.9° C.                              iss                                                                           issupper1ex AAAGGGGATCCATGCAGGATAATA 11 73.6° C.                        AGATGAAAAA                                                                   issupper73ex CACAGGGATCCCAAACGTITACTG 12 77.8° C.                       TTGGAAACAA                                                                   isslower338ex CGCCGGAATTCGCAGATGAGCTCC 13 82.4° C.                      CCATATC                                                                      issupperdiag ATGCAGGATAATAAGATGAAAAA 14 47.6° C.                       isslowerdiag ATAGATGCCAAAAGTGATAAAAC 15 47.2° C.                     __________________________________________________________________________

ompA and iss sequences were amplified according to the followingprocedure. A single colony of an E. coli isolate was transferred into 20μl of Gene Releaser™ (Bioventures, Inc., Murfeesboro, Tenn.) andsubjected to microwaves on the high setting for approximately 6 minutes(Kenmore, ultra-defrost). A PCR master mix consisting of 47.5 μl of H₂O, 10.0 μl of 10×PCR Buffer II (Perkin Elmer, Branchburg, N.J.), 16.0 μlof 1.25 mM dNTP mix (Promega), 0.5 μl of Taq DNA Polymerase (Promega,Madison, Wis.), 1.0 μl of 0.1 mM of each primer, and 4.0 μl of 25 mMMgCl₂ was added to an E. coli-Gene Releaser™ suspension. Amplificationwas performed according to the following parameters: 2 minutes at 97°C.; 1 minute at 97° C.; 1 minute at 49° C., 1 minute at 72° C. for 9cycles; 1 minute at 95° C., 1 minute at 49° C., 1 minute at 72° C. for24 cycles; 5 minutes at 72° C. and then the sample is maintained at 4°C.

Restriction enzyme digest plasmid fragments and amplification productswere separated by horizontal gel electrophoresis. Amplified fragmentswere identified by size, excised from the agarose and purified usingGENECLEAN™ (Bio101, La Jolla, Calif.) or the Wizard PCR Clean-Up System(Promega, Madison, Wis.). The identities of the amplicons were furtherconfirmed by sequencing according to the procedures described below. Toprepare probes, isolated fragments were labeled using a non-radioactive,random-primed DNA labeling kit (Genius I Labeling and Detection Kit,Boehringer Mannheim, Indianapolis, Ind.).

Sequencing of Amplified DNA

Sequencing was performed according to the manufacturer's protocol usingthe LI-COR 4000LR Automated DNA Sequencer (Lincoln, Nebr.). Briefly, 9μl of each purified PCR product, containing 0.1 to 0.6 pmol DNA, wasadded to 2 μl of 1 pmol/μl of the appropriate primer DNA (for example,SEQ ID NOs: 3, 4, 9 and 10 in Table 4) 1.0 μl of dNTP mix, 1.0 μl ofIRD40-dATP at 20 pmol/μl (Boehringer Mannheim, Indianapolis, Ind.), 2.5μl of 10× Sequitherm™ Reaction Buffer (Epicentre Technologies, Madison,Wis.), and 1.5 μl of Sequitherm™ DNA Polymerase at 5 u/μl (Epicentre,Madison, Wis.). All components were mixed carefully in labeled tubes andcovered with mineral oil. The tubes containing this mixture were placedin the thermal controller, and the cycle sequencing reaction was run asdescribed in LI-COR (Lincoln, Nebr.) application bulletin 41.

The first series of cycles were the labeling reactions, wherein anIRD40-DATP was added to the extended primer sequence. The second seriesof cycles was the termination reaction for which 4 μl of the labelingreaction was added to 2 μl of each ddNTP termination mix before cycling.Unincorporated label was removed from the products by ethanolprecipitation according to manufacturer's directions. Samples wereseparated on 4.0% Long Ranger acrylamide gels (FMC, Rockland, Me.) andanalyzed via a LI-COR 4000 LR automated sequencer.

Amplification for Cloning

ompA and iss sequences used for cloning were amplified according to thefollowing procedure. A single colony of E. coli isolate was transferredinto 20 μl of Gene Releaser™ (Bioventures, Inc., Murfeesburo, Tenn.) andsubjected to microwaves on the high setting for approximately 6 minutes(Kenmore, ultra-defrost). Alternatively, a colony was transferred into40 μl SCLB (10 mM Tris-HCl pH 7.5/1 mM EDTA/50 μg/ml proteinase K), andheated at 55° C. for 10 minutes, then heated at 80° C. for 10 minutes,diluted with 80 μl ddH₂ O, cell debris was pelleted and 10 μl of thesupernatant was used.

The prepared E. coli DNA was added to a master mix consisting of47.5-53.5 μl ddH₂ O, 10.0 μl of 10×PCR Buffer II (Perkin Elmer orPromega, Madison, Wis.), 0.5 μl of Amplitaq DNA polymerase (PerkinElmer), 1.0 μl of 0.1 mM of each appropriate primer and 4.0 μl or 8.0 μlof 25 mM MgCl₂.

The amplification cycles were as previously described except that theannealing temperature was 49° C. or 51.8° C. depending on the primerpair used. Amplified fragments for ligation into the expression vectorpGEX-6P-3, were digested with BamHI and EcoRI to produce sticky ends forthe ligation process. Amplified fragments for ligation into pGEM-Tvector have "A" overhangs left by Taq polymerase. Restriction enzymedigest plasmid fragments and amplification products were separated byhorizontal gel electrophoresis. Amplified fragments were identified bysize, excised from the agarose and purified using the Wizard PCRClean-Up System (Promega, Madison, Wis.). T7 DNA ligase was used toligate amplification fragments into the vectors. The identities of theamplicons were further confirmed by sequencing according to theprocedures described below.

Sequencing of Cloned DNA

Sequencing was performed according to the manufacturer's protocol usingthe LI-COR 400OLR Automated DNA Sequencer (Lincoln, Nebr.). Briefly, 9μl of each purified plasmid clone containing 0.2 to 0.6 pmol DNA, wasadded to 1 μl of 1 pmol/μl IRD41 labeled primer DNA (Table 5), 2.5 μl of10× Sequitherm™ Reaction Buffer (Epicentre Technologies, Madison, Wis.),and 1.0 μl of Sequitherm™ DNA Polymerase at 5 u/μl (Epicentre, Madison,Wis.) and 2.5 μl ddH₂ O. All components were mixed carefully before 4 μlwere aliquoted to 2 μl of each ddNTP termination mix in labeled tubesand covered with mineral oil. The tubes containing this mixture wereplaced in the thermal controller, and the cycle sequencing reaction wasrun as described in LI-COR (Lincoln, Nebr.) application bulletin 13.Samples were separated on 4.0% acrylamide gels and analyzed via a LI-COR4000 LR automated sequencer.

                  TABLE 5                                                         ______________________________________                                        Name     Sequences (5' to 3') SEQ ID NO.                                      ______________________________________                                        M13 Forward                                                                            CACGACGTTGTAAAACGAC  16                                                (-29)/IRD41                                                                   Dye-labeled                                                                   primer                                                                        M13 Reverse GGATAACAATTTCACACAGG 17                                           IRD41 Dye-                                                                    labeled primer                                                                5'pGEX GGGCTGGCAAGCCACGTTTGGTG 18                                             IRD41 Dye-                                                                    labeled                                                                       sequencing                                                                    primer                                                                        3'pGEX CCGGGAGCTGCATGTQTCAGAGG 19                                             IRD41 Dye-                                                                    labeled                                                                       sequencing                                                                    primer                                                                      ______________________________________                                    

Colony Blotting

Isolates were stab-inoculated into LB agar and incubated overnight at37° C. Table 3 indicates positive and negative control organisms.Colonies were transferred to charge-modified nylon membranes (QIABRANENylon Plus membrane, QIAGEN, Inc., Chatsworth, Calif.) by the method ofGrunstein and Hogness (Grunstein et al., Proc. Natl. Acad. Sci. (USA),72:3961-3065 (1975)). The colonies were lysed and the DNA denatured.Membranes were then stored and sealed in plastic bags (GibcoBRL,Gaithersburg, Md.) at 4° C.

Total DNA Isolation and Blotting

Total DNA from all test isolates, including non-E. coli strains andcontrol organisms, were isolated by the method of Marmur, J. Mo. Biol.,3:208-218 (1961). Briefly, individual isolates were grown overnight at37° C. with agitation in 50 ml of BHI. Cultures were pelleted bycentrifugation (15 minutes at 5000×g) and the pellets were resuspendedin 5 ml of BPES buffer (10 mM HPO₄, 0.2 M NaCl₂, and 1 mM EDTA, pH 8.0).Five mg of lysozyme (Sigma Chemical Co., St. Louis, Mo.) was added toeach suspension and incubated for 45 minutes at 37° C. with gentleagitation prior to addition of 0.1 ml of 25% SDS and 1.0 ml of 5 Msodium perchlorate (Sigma). Suspensions were then incubated for 15minutes at 65° C. with occasional swirling, cooled to room temperature,and 6.5 ml of chloroform isoamyl alcohol (24:1) (Amresco, Solon, Ohio)was added to each suspension. Tubes were shaken for 5 minutes, and thesuspensions pelleted by centrifugation (10 minutes at 600×g). Theaqueous phase was removed to a glass beaker containing two volumes ofcold absolute ethanol. Sterile glass rods were used to spool the DNA,which was put into 25 ml plastic, screw-top centrifuge tubes (FisherScientific, Chicago, Ill.). These tubes were centrifuged for 10 minutesat 700×g, the ethanol decanted, and the DNA allowed to dry. DNA wasresuspended in sterile 1×TE buffer (100 mM Tris-Cl, 10 mM EDTA pH 8.0)(5 Prime→3 Prime, Inc. Boulder, Colo.).

Samples of total DNA from each organism were spotted onto nylon, thespots allowed to dry, and the DNA denatured in Denaturation Solution (5Prime→3 Prime, Inc., Boulder, Colo.) for 15 minutes. These "dot" blotmembranes were dried briefly on filter paper and were put inNeutralization Solution (5 Prime→3 Prime, Inc., Boulder, Colo.) for 15minutes. Membranes were then placed on filter paper saturated with 2×SSC(pH 7.0; 5 Prime - 3 Prime, Inc., Boulder, Colo.) for 5 minutes. Allfilters were stored in sealed plastic bags (Gibco BRL) at 4° C.

Hybridization Studies

Membranes were prehybridized in aqueous solution (PrehybridizationSolution, 5 Prime→3 Prime, Inc.) for 4 hours at 68° C. and hybridizedwith the individual, denatured probes at 68° C. for 12 hours (Russo etal., Infect. Immun. 61:3578-3582 (1993)). The filters were given two 1hour washes in 0.1×SSC (5 Prime→3 Prime, Inc., Boulder, Colo.) with 0.1%SDS (Sigma Chemical Co., St. Louis, Mo.). Hybridized probes weredetected using the protocol in the Genius 1 kit (Sambrook, Fitsch &Maniatis, Molecular Cloning; A Laboratory Manual, Cold Spring HarborLaboratory Press; pp 9.34-9.55 (1989)).

Capsule Staining

All isolates were examined for the presence of capsule using the methodof Hiss, P. H. Jr., J. Exp. Med. 6:317:345 (1905). Briefly, a loopful ofnormal horse serum was mixed with a loopful of overnight culture on aglass microscopic slide. The film was allowed to air dry and then washeat fixed. The film was covered with crystal violet stain (0.1 g ofcrystal violet per 100 ml of H₂ O) and warmed until steam appeared. Thecrystal violet was removed with 20% (wt/vol) aqueous copper sulfatesolution (CuSO₄, 5H₂ O; Fisher Scientific), blotted dry, the samecovered in oil, a coverslip placed on top of the oil, and examined bylight microscopy.

Biostatistics

To determine if a factor was characteristic of isolates from sick orhealthy birds, the percent positive for each factor in each group wascompared using a Z test of proportions (Maxwell, A. E., AnalyzingQualitative Data (Methuen Co., London (1961)). Also, the correlation ofcvaC with traT- and iss sequences was calculated for the isolates fromsick and healthy birds using the Pearson Product Moment Correlation(Maxwell, A. E., Analyzing Qualitative Data, Methuen Co., London(1961)).

Results

Hybridization Studies

Colony blot hybridization procedures were performed on all test andcontrol isolates with each probe. None of the non-E. coli strains weredetected by any of the prepared probes. Total DNA "dot" blots were foundto yield unambiguous results, although the hybridization results wereoccasionally unclear, for example, false positives or false negativesobtained with the control organisms. The following results were obtainedfrom either unambiguous colony blots or from "dot" blots.

It was observed that there was no significant difference in thedistribution of traT, cvaC, or ompA sequences between the isolates fromsick poultry and healthy poultry. All E. coli isolates contained ompAsequences. There was, however, a highly significant difference in thedistribution of iss sequences in the isolates of sick poultry andhealthy poultry, (p<0.000000001) as the isolates from sick poultry weremuch more likely to contain iss sequences than were the isolates fromhealthy poultry as shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        % isolates detected with gene                                                 Gene  Sick Birds                                                                              Healthy Birds                                                                             Z     Probability of Z                            ______________________________________                                        cvaC  66.66     58.9        1.12  0.26                                          traT 72.7 73.33 0.34 0.73                                                     iss 76.33 23.2 6.69 0.000000001                                               capsule 1.9 8.9 2.57 0.01                                                   ______________________________________                                    

Few encapsulated E. coli isolates were detected, and statisticalanalysis indicated that E. coli isolates from healthy poultry were morelikely to be encapsulated than were the E. coli isolates from sickpoultry (p<0.01, see Table 6).

Correlations between the occurrence of related sequences within theisolates from healthy or sick poultry were also determined for genesreportedly linked together on ColV plasmids (Waters et al., Microbiol.Rev., 55:437-450 (1991)). For example, the occurrence of cvaC-relatedsequences for the E. coli isolates from healthy and sick poultry was notsignificantly correlated with the occurrence of iss-or traT-relatedsequences.

Approximately 23% of the non-disease-associated avian E. coli isolatesexamined contained iss-related sequences. It appears the iss isolates ofhealthy poultry are capable of causing disease, or that the isolates donot express the Iss protein or export the Iss polypeptide to the outermembrane where it is active in protection of the bacterium against hostcomplement.

Detection of Iss on the Surface of Avian E. coli Isolates

Several methods are available to determine if an E. coli isolateexpresses Iss. A preferred method indicates the presence of Iss on theouter membrane of the host bacterium where Iss can exert itsanti-complement effect. Probing RNA using Northern blotting techniquesprovides evidence of iss transcription, but not translation or presenceof Iss in the outer membrane. ELISAs to detect Iss in bacterial lysatesare easy to perform and indicate the presence of Iss, but are not usefulin determining whether ISS is present on the bacterial outer membrane.Assays useful to determine the location of Iss includefluorescent-antibody techniques or flow cytometry. Preferably, flowcytometry is employed to detect the location of Iss protein due to theaccuracy and quantitative nature of the data obtained by this method.

To prepare samples for analysis by flow cytometry, bacteria are grown inBrain Heart Infusion (BHI) broth for 18 hours at 37° C. Bacteria arethen washed and resuspended in buffer. Monoclonal antibodies specificfor Iss are added to each bacterial suspension and allowed to incubatefor 30 minutes. Suspensions are pelleted, washed thoroughly, andincubated with FITC-labeled, goat anti-mouse Ig conjugated antibody for30 minutes at 0° C. After incubation, suspensions are pelleted, washedthoroughly, fixed with paraformaldehyde, and analyzed by flow cytometryusing a FALSCalibur (Becton Dickinson, San Jose, Calif.) (Otten et al.,Flow Cytometry Analysis Using the Becton Dickinson FACScan, John Wiley &Sons, New York, pp. 5.4.1-5.4.19 (1992)).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 22                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 760 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GTGGCGAAAA CTAGTAAAAC AGCAACCCGA ACCACTTGAT GTGCATCGTT TT -            #TGATTATT     60                                                                 - - CCCGTATACT CTTGCAGAAG GAGTTCTCCG TCGGGCTACT GTCATGGTTA AT -            #GCGGGGAA    120                                                                 - - TATGGCGACA ATACAACACA CCTAAAAGAG TAATGGACAG ATGAAGCGGT TT -            #ATTCATTT    180                                                                 - - CCCATGATTC TGAGTACCTA CCAAGTCTGA GTAACCACTT TTATACTTTT AA -            #TTTTCGTT    240                                                                 - - CATTTAGCTA TCGTTTAATT ATTATCACAT AGGATTCTGC CGTTTTTAAC AA -            #TGCAGGAT    300                                                                 - - AATAAGATGA AAAAAATGTT ATTTTCTGCC GCTCTGGCAA TGCTTATTAC AG -            #GATGTGCT    360                                                                 - - CAACAAACGT TTACTGTTGG AAACAAACCG ACAGCAGTAA CACCAAAGGA AA -            #CCATCACT    420                                                                 - - CATCATTTCT TCGTTTCGGG AATTGGACAA GAGAAAACTG TTGATGCAGC CA -            #AAATTTGT    480                                                                 - - GGCGGTGCAG AAAATGTTGT TAAAACAGAA ACTCAGCAAA CATTCGTAAA TG -            #GATTGCTC    540                                                                 - - GGTTTTATCA CTTTTGGCAT CTATACTCCG CTGGAAGCCC GGGTATATTG CT -            #CACAATAG    600                                                                 - - TTGCCCATCG ATATGGGGAG CTCATCTGCA CTGTTCATTA ATATACTTCT GG -            #GCTCCCTA    660                                                                 - - CAGTTGTTTT TGCATAGTGA TAAGCCTCTC TCTGAGGGAG GAAATAATCC TG -            #TTCAGCGA    720                                                                 - - TGTCTGCCAG TCGGGGGGCT GCATTATCCA CCCCGAGGCG     - #                      - #   760                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Gln Asp Asn Lys Met Lys Lys Met Leu Ph - #e Ser Ala Ala Leu Ala      1               5   - #                10  - #                15               - - Met Leu Ile Thr Gly Cys Ala Gln Gln Thr Ph - #e Thr Val Gly Asn Lys                  20      - #            25      - #            30                   - - Pro Thr Ala Val Thr Pro Lys Glu Thr Ile Th - #r His His Phe Phe Val              35          - #        40          - #        45                       - - Ser Gly Ile Gly Gln Glu Lys Thr Val Asp Al - #a Ala Lys Ile Cys Gly          50              - #    55              - #    60                           - - Gly Ala Glu Asn Val Val Lys Thr Glu Thr Gl - #n Gln Thr Phe Val Asn      65                  - #70                  - #75                  - #80        - - Gly Leu Leu Gly Phe Ile Thr Phe Gly Ile Ty - #r Thr Pro Leu Glu Ala                      85  - #                90  - #                95               - - Arg Val Tyr Cys Ser Gln                                                              100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GTGGCGAAAA CTAGTAAAAC AGC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CGCCTCGGGG TGGATAA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - ATGCAGGATA ATAAGATGAA AAAAATGTTA TTTTCTGCCG CTCTGGCAAT GC -             #TTATTACA     60                                                                 - - GGATGTGCTC AACAAACGTT TACTGTTGGA AACAAACCGA CAGCAGTAAC AC -            #CAAAGGAA    120                                                                 - - ACCATCACTC ATCATTTCTT CGTTTCCCCA ATTGGACAGA GAAAACTGTT GA -            #TGCAGCCA    180                                                                 - - AAATTTGTTG GCGGTGCAGA AAATGTTGTT AAAACAGAAA CTCAGCAAAC AT -            #TCGTAAAT    240                                                                 - - GCATTGCCCG GTTTTATCAC TTTTGGCATC TATACTCCGC GGGAAACCCG TG -            #TATATTGC    300                                                                 - - TCACAATAG                - #                  - #                      - #        309                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - ATCGGGAATA ACACCATGAA AAAAATGCTA CTCGCTACTG CGCTGGCCCT GC -             #TTATTACA     60                                                                 - - GGATGTGCTC AACAGACGTT TACTGTTCAA AACAAACCGG CAGCAGTAGC AC -            #CAAAGGAA    120                                                                 - - ACCATCACCC ATCATTTCTT CGTTTCTGGA ATTGGGCAGA AGAAAACTGT CG -            #ATGCAGCC    180                                                                 - - AAAATTTGTG GCGGCGCAGA AAATGTTGTT AAAACAGAAA CCCAGCAAAC AT -            #TCGTAAAT    240                                                                 - - GGATTGCTCG GTTTTATTAC TTTAGGCATT TATACTCCGC TGGAAGCGCG TG -            #TGTATTGC    300                                                                 - - TCACAATAA                - #                  - #                      - #        309                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Met Gln Asp Asn Lys Met Lys Lys Met Leu Ph - #e Ser Ala Ala Leu Ala      1               5   - #                10  - #                15               - - Met Leu Ile Thr Gly Cys Ala Gln Gln Thr Ph - #e Thr Val Gly Asn Lys                  20      - #            25      - #            30                   - - Pro Thr Ala Val Thr Pro Lys Glu Thr Ile Th - #r His His Phe Phe Val              35          - #        40          - #        45                       - - Ser Pro Ile Gly Gln Arg Lys Leu Leu Met Gl - #n Pro Lys Phe Val Gly          50              - #    55              - #    60                           - - Gly Ala Glu Asn Val Val Lys Thr Glu Thr Gl - #n Gln Thr Phe Val Asn      65                  - #70                  - #75                  - #80        - - Ala Leu Pro Gly Phe Ile Thr Phe Gly Ile Ty - #r Thr Pro Arg Glu Thr                      85  - #                90  - #                95               - - Arg Val Tyr Cys Ser Gln                                                              100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Lys Lys Met Leu Leu Ala Thr Ala Leu Al - #a Leu Leu Ile Thr Gly      1               5   - #                10  - #                15               - - Cys Ala Gln Gln Thr Phe Thr Val Gln Asn Ly - #s Pro Ala Ala Val Ala                  20      - #            25      - #            30                   - - Pro Lys Glu Thr Ile Thr His His Phe Phe Va - #l Ser Gly Ile Gly Gln              35          - #        40          - #        45                       - - Lys Lys Thr Val Asp Ala Ala Lys Ile Cys Gl - #y Gly Ala Glu Asn Val          50              - #    55              - #    60                           - - Val Lys Thr Glu Thr Gln Gln Thr Phe Val As - #n Gly Leu Leu Gly Phe      65                  - #70                  - #75                  - #80        - - Ile Thr Leu Gly Ile Tyr Thr Pro Leu Glu Al - #a Arg Val Tyr Cys Ser                      85  - #                90  - #                95               - - Gln                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CTTGCGGAGG CTTGTCTGAG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - AGGCATTGCT GGGTAAGGAA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AAAGGGGATC CATGCAGGAT AATAAGATGA AAAA       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - CACAGGGATC CCAAACGTTT ACTGTTGGAA ACAA       - #                  -     #        34                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - CGCCGGAATT CGCAGATGAG CTCCCCATAT C        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - ATGCAGGATA ATAAGATGAA AAA           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - ATAGATGCCA AAAGTGATAA AAC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CACGACGTTG TAAAACGAC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - GGATAACAAT TTCACACAGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - GGGCTGGCAA GCCACGTTTG GTG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - CCGGGAGCTG CATGTGTCAG AGG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Se - #r Met Gln Asp Asn Lys      1               5   - #                10  - #                15               - - Met Lys Lys Met Leu Phe Ser Ala Ala Leu Al - #a Met Leu Ile Thr Gly                  20      - #            25      - #            30                   - - Cys Ala Gln Gln Thr Phe Thr Val Gly Asn Ly - #s Pro Thr Ala Val Thr              35          - #        40          - #        45                       - - Pro Lys Glu Thr Ile Thr His His Phe Phe Va - #l Ser Gly Ile Gly Gln          50              - #    55              - #    60                           - - Glu Lys Thr Val Asp Ala Ala Lys Ile Cys Gl - #y Gly Ala Glu Asn Val      65                  - #70                  - #75                  - #80        - - Val Lys Thr Glu Thr Gln Gln Thr Phe Val As - #n Gly Leu Leu Gly Phe                      85  - #                90  - #                95               - - Ile Thr Phe Gly Ile Tyr Thr Pro Leu Glu Al - #a Arg Val Tyr Cys Ser                  100      - #           105      - #           110                  - - Gln                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 378 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - CTGGAAGTTC TGTTCCAGGG GCCCCTGGGA TCCATGCAGG ATAATAAGAT GA -             #AAAAAATG     60                                                                 - - TTATTTTCTG CCGCTCTGGC AATGCTTATT ACAGGATGTG CTCAACAAAC GT -            #TTACTGTT    120                                                                 - - GGAAACAAAC CGACAGCAGT AACACCAAAG GAAACCATCA CTCATCATTT CT -            #TCGTTTCG    180                                                                 - - GGAATTGGAC AAGAGAAAAC TGTTGATGCA GCCAAAATTT GTGGCGGTGC AG -            #AAAATGTT    240                                                                 - - GTTAAAACAG AAACTCAGCA AACATTCGTA AATGGATTGC TCGGTTTTAT CA -            #CTTTTGGC    300                                                                 - - ATCTATACTC CGCTGGAAGC CCGGGTATAT TGCTCACAAT AGTTGCCCAT CG -            #ATATGGGG    360                                                                 - - AGCTCATCTG CGAATTCC             - #                  - #                      - # 378                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - ATGCAGGATA ATAAGATGAA AAAAATGTTA TTTTCTGCCG CTCTGGCAAT GC -             #TTATTACA     60                                                                 - - GGATGTGCTC AACAAACGTT TACTGTTGGA AACAAACCGA CAGCAGTAAC AC -            #CAAAGGAA    120                                                                 - - ACCATCACTC ATCATTTCTT CGTTTCGGGA ATTGGACAAG AGAAAACTGT TG -            #ATGCAGCC    180                                                                 - - AAAATTTGTG GCGGTGCAGA AAATGTTGTT AAAACAGAAA CTCAGCAAAC AT -            #TCGTAAAT    240                                                                 - - GGATTGCTCG GTTTTATCAC TTTTGGCATC TATACTCCGC TGGAAGCCCG GG -            #TATATTGC    300                                                                 - - TCACAATAG                - #                  - #                      - #        309                                                                __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleic acid sequence that encodes an E. coli Iss polypeptide whereinthe nucleic acid molecule is isolated from E. coli obtained from a bird.2. The nucleic acid molecule of claim 1 wherein the nucleic acidsequence comprises SEQ ID NO.
 1. 3. The nucleic acid molecule of claim 1wherein the nucleic acid sequence encodes a polypeptide having an aminoacid sequence comprising SEQ ID NO.
 2. 4. The nucleic acid molecule ofclaim 1 wherein the nucleic acid sequence is operably linked to apromoter functional in a host cell so as to form an expression vector.5. An expression vector comprising the nucleic acid molecule of claim 1further comprising control sequences recognized by a host celltransformed with the nucleic acid.
 6. A method of using a nucleic acidmolecule encoding an E. coli Iss polypeptide, the method comprisingexpressing the nucleic acid molecule of claim 1 in a cultured host cellstably transformed with a vector comprising the nucleic acid sequenceoperably linked to control sequences recognized by the host cell.
 7. Themethod of claim 6 which further comprises recovering the polypeptidefrom the host cell.
 8. The method of claim 6 wherein the nucleic acidmolecule comprises SEQ ID NO.
 1. 9. The method of claim 6 wherein thepolypeptide has an amino acid sequence comprising SEQ ID NO.
 2. 10. Amethod for producing a recombinant E. coli Iss polypeptidecomprising:(a) providing an expression vector that comprises a nucleicacid sequence according to claim 1 operably linked to control sequencesrecognized by a host cell; (b) transforming the host cell with thevector; and (c) culturing the resultant transformed cell underconditions that allow expression of the recombinant polypeptide encodedby the nucleic acid sequence.
 11. A method according to claim 10 whereinthe host cell is prokaryotic.
 12. A method according to claim 11 whereinthe prokaryotic host cell is a gram negative or gram positive organism.13. A method according to claim 11 wherein the host cell is E. coli. 14.A method according to claim 10 wherein the host cell is eukaryotic. 15.The isolated nucleic acid molecule of claim 1 wherein the bird issuspected of being exposed to, a carrier of, or afflicted with asepticemic disease.
 16. The isolated nucleic acid molecule of claim 1wherein the presence of the nucleic acid molecule is associated with asepticemic disease in the bird.
 17. The nucleic acid molecule of claim 1wherein the nucleic acid sequence comprises SEQ ID NO:
 22. 18. Themethod of claim 6 wherein the nucleic acid molecule comprises SEQ ID NO:22.
 19. The method of claim 10 wherein the nucleic acid sequencecomprises SEQ ID NO: 22.